Angiotensin-(1-7) and angiotensin-(1-7) agonists for inhibition of cancer cell growth

ABSTRACT

The present invention describes the use of angiotensin-(1-7) peptide as an anti-cancer therapeutic. Thus, in one embodiment, the present invention comprises a composition to inhibit the growth of cancer cells in an individual comprising a pharmaceutically effective amount of an agonist for the angiotensin-(1-7) receptor to inhibit cancer cell growth or proliferation. Application of a pharmaceutically effective amount of angiotensin-(1-7) or angiotensin-(1-7) receptor agonist is associated with an increase in the expression of genes involved in tumor suppression, apoptosis, and/or cell cycle inhibition, and a decrease the expression of known oncogenes, protein kinases, and/or cell cycle progression genes. Cancers treated using the methods and compositions described herein include cancers having an angiotensin-(1-7) receptor, including, but not limited to, breast and lung cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/359,847, filed Feb. 27, 2002. The disclosure of U.S.Provisional Application Serial No. 60/359,847 is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions and methods for thetreatment and prevention of cancer. More specifically, the presentinvention relates to the use of angiotensin-(1-7) or other agonists forthe angiotensin-(l1-7) receptor as anticancer therapeutics.

BACKGROUND

[0003] Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide hormonewhich is normally present in the circulation at concentrations similarto angiotensin II (Ang II) and is primarily derived from angiotensin I(Ang I) by tissue peptidases, including neprilysin, thimetoligopeptidase and prolyl endopeptidase (Ferrario, C. M. et al.,Hypertension, 1997, 30:535-541) and by angiotensin converting enzyme(ACE) 2 from angiotensin II (Ang II) (Vickers, C., et al., J. Biol.Chem., 2002, 277:14836-14843). In addition, Ang-(1-7) is a substrate forACE (Chappell, M. C. et al. Hypertension, 1998, 31:362-367). ACEcatalyzes the conversion of angiotensin I (Ang I) to the biologicallyactive peptide angiotensin II [Ang II]. Treatment of patients or animalswith ACE inhibitors results in a significant elevation in thecirculating and tissue levels of Ang II, as well as the N-terminalheptapeptide fragment of Ang II, angiotensin-(1-7) (Campbell, M. C. etal., Hypertension, 1993, 22:513-522; Kohara, K. et al., Hypertension,1991, 17:131-138; Lawrence, A. C. et al., J. Hypertens., 1990,8:715-724; and Luque, M. et al., J. Hypertens., 1996, 14:799-805). Ithas been suggested that ACE inhibition not only elevates Ang-(1-7) byincreasing Ang I, the substrate for Ang-(1-7) production, but also bypreventing Ang-(1-7) conversion to the inactive fragment Ang-(1-5).

[0004] Although Ang-(1-7) was long-considered an inactive product of thedegradation of Ang II, studies showed that the heptapeptide producesunique physiological responses which are often opposite to those of thewell-recognized angiotensin peptide, Ang II (Ferrario, C. M. et al.,Hypertension, 1997, 30:535-541). Thus, Ang-(l-7) has been shown tostimulate vasopressin release from neuropeptidergic neurons (Schiavone,M. T. et al., Proc. Natl. Acad. Sci. USA, 1988, 85:4095-4098), increasethe release of certain neurotransmitters (Ambuhl, P. et al., Regul.Pept., 1992, 38:111-120), reduce blood pressure in hypertensive dogs andrats (Benter, I. F. et al., Am J. Physiol. Heart Circ. Physiol., 1995,269:H313-H319; and Nakamoto, H. et al., Hypertension, 1995, 25:796-802),and have biphasic effects on renal fluid absorption (DelliPizzi, A. etal., Br. J. Pharmacol., 1994, 111:1-3; DelliPizzi, A. et al.,Pharmacologist, 34, 1992; Garcia, N. H. and Garbin, J. L., J. Am. Soc.Nephrol., 1994, 5:1133-1138; Handa, R. K. et al., Am. J. Physiol., 1996,270:F141-F147; and Hilchey, S. D. and Bell-Quilley, C. P., Hypertension,1995, 25:1238-1244).

[0005] Besides its role in reducing blood pressure, Ang-(1-7) attenuatesvascular growth both in vitro and in vivo (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108; Strawn, W. B. et al., Hypertension,1999, 33:207-211; and Tallant, E. A. et al., Hypertension, 1999,34:950-957). Also, hypertensive patients administered ACE inhibitorsshow a reduced risk of cancer, particularly lung and sex-specificcancers (Jick, H., et al., Lancet, 1997, 349:525-528; Lever, A. F. etal., Lancet, 1998, 352:179-184; and Pahor, M. et al., Am. J. Hypertens.,1996, 9:695-699).

[0006] What is needed in cancer prevention and therapeutics is a way toprevent tumors from forming, or to inhibit the growth of tumors onceformed. Also, what is needed are agents that act specifically at thetumor cell, thus minimizing non-specific and/or toxic side effects.Preferably, the chemotherapeutic agents will comprise ligands thattarget the chemotherapeutic agent to cancer cells with high efficacy toeither reduce cellular signals that promote cell growth, or to increasecellular signals that promote cell death.

SUMMARY OF THE INVENTION

[0007] The present invention relates to the use of angiotensin-(1-7)[Ang-(1-7)] receptor agonists as anticancer therapeutics. Thus,embodiments of the present invention describes the use of agonists forthe Ang-(1-7) receptor, such as the Ang-(1-7) peptide and derivativesthereof, or agents which increase levels of plasma, tissue or cellularAng- (1-7), as compounds for prevention and treatment of cancer cellgrowth and proliferation.

[0008] Embodiments of the present invention recognize that Ang-(1-7) caninhibit tumor cell growth in vitro and in vivo. Preferably, cancerstreated by the method of the present invention comprise bladder cancer,breast cancer, brain cancer, colon cancer, endometrial cancer, head andneck cancer, leukemia, lymphoma, lung cancer, melanoma, liver cancer,rectal cancer, ovarian cancer, prostate cancer, bone cancer, pancreaticcancer, skin cancer, or renal cancer.

[0009] In one embodiment, the present invention comprises a compositionfor inhibition of cell growth or proliferation comprising apharmaceutically effective amount of an agonist for theangiotensin-(1-7) receptor in a pharmaceutically acceptable carrier,wherein a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist comprises an amount which is sufficient to inhibit cellgrowth or proliferation.

[0010] In an embodiment, the present invention comprises a compositionfor inhibition of cancer cell growth or proliferation comprising apharmaceutically effective amount of an agonist for theangiotensin-(1-7) receptor in a pharmaceutically acceptable carrier,wherein a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist inhibits growth or proliferation of the cancer cells.In an embodiment, the angiotensin-(1-7) receptor agonist comprisesangiotensin-(1-7) peptide having the sequence set forth in SEQ ID NO: 1.

[0011] Another embodiment of the present invention comprises acomposition to inhibit the growth or proliferation of cancer cells in anindividual comprising a pharmaceutically effective amount of a compoundwhich provides sufficient angiotensin-(1-7) receptor agonist to inhibitgrowth or proliferation of the cancer cells.

[0012] In one embodiment, the present invention comprises a method toinhibit cell growth or proliferation comprising application an agonistfor the angiotensin-(1-7) receptor to the cells, wherein the cells havea functional angiotensin-(l-7) receptor.

[0013] In another embodiment, the present invention comprises a methodto inhibit the growth or proliferation of cancer cells in an individualcomprising application of a pharmaceutically effective amount of anagonist for the angiotensin-( 1-7) receptor to the individual, wherein apharmaceutically effective amount comprises sufficient angiotensin-(l-7) receptor agonist to inhibit growth or proliferation of the cancercells.

[0014] In yet another embodiment, the present invention comprises amethod to inhibit the growth or proliferation of cancer cells in anindividual comprising application of a pharmaceutically effective amountof a compound which increases the efficacy or amount of circulating orcellular angiotensin-(1-7) receptor agonist.

[0015] In yet another embodiment, the present invention comprises a kitfor inhibiting cancer cell growth and proliferation in an individualcomprising: (a) at least one container comprising a pharmaceuticallyeffective amount of a functional agonist for the angiotensin-(l -7)receptor; (b) a pharmaceutically acceptable carrier; and (c)instructions for use.

[0016] From the foregoing summary, it is apparent that an object of thepresent invention is to provide methods and compositions for the use ofangiotensin-(l1-7) receptor agonists as anti-cancer therapeutics. Thereare, of course, additional features of the invention which will bedescribed hereinafter and which will form the subject matter of theclaims appended hereto. It is to be understood that the invention is notlimited in its application to the specific details as set forth in thefollowing description and figures. The invention is capable of otherembodiments and of being practiced or carried out in various ways.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 shows the dose-dependent effect of angiotensin peptides on3H-thymidine incorporation into vascular smooth muscle cells (VSMCs) inaccordance with an embodiment of the present invention.

[0018]FIG. 2 shows inhibition of the 1 μM Ang-(1-7)(Asp—Arg—Val—Tyr—Ile—His—Pro) (SEQ ID NO: 1) mediated reduction (+A7) inserum-stimulated growth (FBS) by [Sar¹—Thr⁸]-Ang II (Sarthran)(Sar—Arg—Val—Tyr—Ile—His—Pro—Thr) (SEQ ID NO: 2) or [D—Ala⁷]-Ang-(1-7)(DAlaA7) (Asp—Arg—Val—Tyr—Ile—His—[D]Ala) (SEQ ID NO: 3) but not by AT₁(L158,809) or AT₂ (PD123177) receptor antagonists in accordance with anembodiment of the present invention.

[0019]FIG. 3 shows stained sections of: an uninjured rat carotid artery;a saline-treated injured carotid artery; and an injured carotid arterytreated with Ang-(1-7), in accordance with an embodiment of the presentinvention.

[0020]FIG. 4 shows morphometric analysis of intima and media of injuredrat carotid arteries and the media of uninjured rat carotid arteries inballoon catheter-injured rats infused with either saline or Ang-(1-7)(*P<0.05; n=8) in accordance with an embodiment of the presentinvention.

[0021]FIG. 5 shows that Ang-(1-7) causes a dose-dependent reduction inserum-stimulated ³H-thymidine incorporation into SK-LU-1, A549, andSK-MES-1 human lung cancer cells and ZR-75-1 human breast cancer cells(n=4-8, in triplicate) in accordance with an embodiment of the presentinvention.

[0022]FIG. 6 shows a time-dependent reduction in ³H-thymidineincorporation into SK-LU-1, A549, and SK-MES-1 lung cancer cells andZR-75-1 breast cancer cells in the presence of 100 nM Ang-(l-7) (n=3-4,in triplicate) in accordance with an embodiment of the presentinvention.

[0023]FIG. 7 shows that the Ang-(1-7)-stimulated reduction in³H-thymidine incorporation into SK-LU-1 lung cancer cells is blocked bypretreatment with [D—Ala⁷]-Ang-(1-7) (DalaA7), but not by an AT₁(Losartan) or AT₂ (PD123177) receptor antagonist (n=3, in triplicate) inaccordance with an embodiment of the present invention.

[0024]FIG. 8 shows that Ang-(1-7) (Asp—Arg—Val—Tyr—Ile—His—Pro) (SEQ IDNO: 1), at 1 or 100 nM, reduced serum-stimulated ³H-thymidineincorporation into SK-LU-1 lung cancer cells while Ang I(Asp—Arg—Val—Tyr—Ile—His—Pro—Phe—His—Leu) (SEQ ID NO: 4), Ang-(2-8)(Arg—Val—Tyr—Ile—His—Pro—Phe) (SEQ ID NO: 5), Ang-(3-8)(Val—Tyr—Ile—His—Pro—Phe) (SEQ ID NO: 6), Ang-(3-7)(Val—Tyr—Ile—His—Pro) (SEQ ID NO: 7) and Ang II(Asp—Arg—Val—Tyr—Ile—His—Pro—Phe) (SEQ ID NO: 8) were ineffective (n=3-9in triplicate; * indicates p<0.05) in accordance with an embodiment ofthe present invention.

[0025]FIG. 9 shows inhibition of breast cancer tumor growth by Ang-(1-7)in accordance with an embodiment of the present invention. Tumor-bearingmice infused for 28 days with Ang-(1-7) (n=4) had a 40% reduction intumor size, while the tumors of saline-treated animals (n=3) doubled, ascompared to tumor volume prior to treatment.

[0026]FIG. 10 shows the effect of the cAMP-dependent protein kinaseinhibitor (PKAI) Rp-cAMPS (10 ,M) on the inhibition of serum-stimulated³H-thymidine incorporation by either 1 μM Ang-(1-7) or 5 pM carbacyclinin VSMCs in accordance with an embodiment of the present invention. Theresults are from VSMCs from 3 Sprague Dawley rats and each point was intriplicate.

[0027]FIG. 11 shows that increasing concentrations of Ang-(1-7) causes adose-dependent reduction in ERKI and ERK 2 activities stimulated by 100nM Ang II (n=VSMCs from 7 different rat aortas; * denotes p <0.05), inaccordance with an embodiment of the present invention.

[0028]FIG. 12 shows that Ang-(1-7) causes a dose dependent reduction inserum- stimulated activation of ERKl and ERK2 in SK-LU-1 lung cancercells in accordance with an embodiment of the present invention. Thedata is representative of experiments with SK-LU-1 cells of 3 differentpassage numbers.

[0029]FIG. 13 shows that Ang-(1-7) inhibits platelet-derived growthfactor (PDGF) or epidermal growth factor (EGF)-stimulated ³H-thymidineincorporation into human ZR- 75-1 breast cancer cells in accordance withan embodiment of the present invention.

[0030]FIG. 14 shows a histogram of SK-LU-1 cells treated with Ang-(1-7)in accordance with an embodiment of the present invention. QuiescentSK-LU-1 lung cancer cells were stimulated with 1% FBS for 2 h in thepresence or absence of 100 nM Ang-(1-7). Radiolabeled CDNA, preparedfrom DNase-treated total RNA, was incubated with Human Cancer Atlas cDNAExpression Array (Clontech Laboratories).

[0031]FIG. 15 shows regulation of MEK 5 mRNA and protein by Ang-(1-7) inSK-LU-1 lung cancer cells in accordance with an embodiment of thepresent invention.

[0032]FIG. 16 shows that Ang-(1-7) stimulates apoptosis inmitogen-stimulated SK-LU-1 lung cancer cells as evidenced by an increasein the caspase-3 cleavage product poly(ADP-ribose) polymerase (PARP) asmeasured using an antibody specific to cleaved PARP in serum stimulatedSK-LU-1 cells treated for either 2, 4, or 8 h with 10 nM Ang-(1-7) inaccordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention describes the use of angiotensin-(1-7)receptor agonists, such as angiotensin-(1-7) [Ang-(1-7)](Asp—Arg—Val—Tyr—Ile—His—Pro) (SEQ ID NO: 1) as anticancer therapeutics.Thus, embodiments of the present invention recognize that agonists ofthe Ang-(1-7) receptor can inhibit tumor cell growth in vitro and invivo.

[0034] In one embodiment, the present invention comprises a method toinhibit cell growth or proliferation comprising application of anagonist for the angiotensin-(1-7) receptor to the cells wherein thecells have a functional angiotensin-(1-7) receptor.

[0035] The receptor may be located in either the membrane or within thecellular compartments. Preferably, the cells comprise cancer cells. Morepreferably, the cancer comprises bladder cancer, breast cancer, braincancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.

[0036] In an embodiment, the angiotensin-(1-7) receptor agonistcomprises angiotensin- (1-7) peptide having the sequence set forth inSEQ ID NO: 1. In an embodiment, the angiotensin-(1-7) receptor agonistis modified to increase its chemical stability in vivo. In an alternateembodiment, the angiotensin-(1-7) receptor agonist comprises a fragmentof angiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions, wherein conservativeamino acid substitutions are those substitutions which do notsignificantly effect the structure or function of the peptide. In yetanother embodiment, the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.

[0037] In yet another embodiment, the present invention comprises amethod to inhibit the growth or proliferation of cancer cells in anindividual comprising application of a pharmaceutically effective amountof an agonist for the angiotensin-(1-7) receptor to the individual,wherein a pharmaceutically effective amount comprises sufficientangiotensin-(1-7) receptor agonist to inhibit growth or proliferation ofthe cancer cells. Preferably, the individual is human.

[0038] Preferably, the cancer comprises cells having a functionalangiotensin-(1-7) receptor. The receptor may be on the cell membrane orintracellular. Also preferably, the cancer comprises bladder cancer,breast cancer, brain cancer, colon cancer, endometrial cancer, head andneck cancer, leukemia, lymphoma, lung cancer, melanoma, liver cancer,rectal cancer, ovarian cancer, prostate cancer, bone cancer, pancreaticcancer, skin cancer, or renal cancer.

[0039] In an embodiment, the angiotensin-(1-7) receptor agonistcomprises angiotensin- (1-7) peptide having the sequence set forth inSEQ ID NO: 1. In an embodiment, the angiotensin-(1-7) receptor agonistis modified to increase its chemical stability in vivo. In an alternateembodiment, angiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions, wherein conservativeamino acid substitutions are those substitutions which do notsignificantly effect the structure or function of the peptide. In yetanother embodiment, the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.

[0040] Also preferably, the method includes application of a compoundwhich increases the efficacy or amount of circulating or cellularangiotensin-(1-7) agonist. For example, in an embodiment, the methodincludes application of a compound that increases angiotensin-(1-7)synthesis. Alternatively, the method includes application of a compoundthat decreases angiotensin-(1-7) agonist degradation. In an embodiment,the method includes application of a compound that is an antagonist ofother, non- angiotensin-(1-7) receptor subtypes, such as an anatagonistfor the AT₁ angiotensin receptor.

[0041] Also preferably, application of a pharmaceutically effectiveamount of angiotensin-(1-7) receptor agonist in the individual increasescellular prostacyclins. Also preferably, application of apharmaceutically effective amount of angiotensin-(1-7) receptor agonistin the individual increases cellular cAMP.

[0042] In an embodiment, application of a pharmaceutically effectiveamount of angiotensin-(1-7) receptor agonist increases the expression ofgenes involved in tumor suppression, apoptosis, and/or cell cycleinhibition in the cancer cells. Preferably, the genes showing increasedexpression comprise BAD, oncostatin M-specific beta subunit, PDCD2, EGFresponse factor 1, CASP4, RBQ-3, p16-INK, menin, checkpoint suppressor1, BAK, apoptotic protease activating factor-l, SOCS-3, insulin-likegrowth factor binding protein 2, B-myb or the fau tumor suppressor.

[0043] Alternatively, or additionally, application of a pharmaceuticallyeffective amount of angiotensin-(1-7) receptor agonist in the individualmay also decrease the levels of known oncogenes, protein kinases, and/orcell cycle progression genes in the cancer cells. Preferably, the genesshowing decreased expression comprise cell cycle entry regulator, ERKI,cell cycle progression 2 protein, p21/K-ras 2B oncogene, epithelial cellkinase, ser/thr kinase, MAP kinase kinase 5 (MEK5), beta catenin,tyrosine-protein kinase receptor tyro3 precursor, protein phosphatase 2AB56-alpha, cyclin-dependent kinase regulatory subunit (CDC28), celldivision protein kinase 6 (CDK6), c-myc oncogene, ERBB-3 receptorprotein tyrosine kinase, A-kinase anchoring protein, or rho C.

[0044] In an embodiment, there is a discrete dosage range ofangiotensin-(1-7) receptor agonist which is effective in inhibitingtumor cell growth. Preferably, the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-(1-7) receptoragonist at the cancer which ranges from 0.005 nM to 10 μM. Morepreferably, the dose of angiotensin-(1-7) receptor agonist results in alocal concentration of angiotensin-(1-7) receptor agonist at the cancerwhich ranges from 0.05 nM to 1 μM. Even more preferably, the dose ofangiotensin-(1-7) receptor agonist results in a local concentration ofangiotensin-(1-7) or angiotensin-(1-7) receptor agonist at the cancerwhich ranges from 1 nM to 100 nM.

[0045] In yet another aspect, the present invention comprises a methodto inhibit the growth or proliferation of cancer cells in an individualcomprising application of a pharmaceutically effective amount of acompound to the individual which increases the efficacy or amount ofcirculating or cellular angiotensin-(1-7) agonist.

[0046] In an embodiment, the compound which increases the efficacy oramount of cellular angiotensin-(1-7) agonist increases angiotensin-(1-7)synthesis. In another embodiment, the compound which increases theefficacy or amount of cellular angiotensin-(1-7) agonist may decreaseangiotensin-(1-7) agonist degradation, metabolism or clearance. In yetanother embodiment, the compound which increases the efficacy or amountof cellular angiotensin-(1-7) agonist comprises an angiotensin AT₁receptor antagonist. Preferably, the cancer treated by the method of thepresent invention comprises bladder cancer, breast cancer, brain cancer,colon cancer, endometrial cancer, head and neck cancer, leukemia,lymphoma, lung cancer, melanoma, liver cancer, rectal cancer, ovariancancer, prostate cancer, bone cancer, pancreatic cancer, skin cancer, orrenal cancer.

[0047] In another aspect, the present invention comprises a compositionfor inhibition of cell growth or proliferation comprising apharmaceutically effective amount of an agonist for theangiotensin-(1-7) receptor in a pharmaceutically acceptable carrier,wherein a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist comprises an amount which is sufficient to inhibit cellgrowth or proliferation. Preferably, the cells comprise cancer cells.More preferably, the cancer comprises bladder cancer, breast cancer,brain cancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.

[0048] In another aspect, the present invention comprises a compositionfor inhibition of cancer cell growth or proliferation comprising apharmaceutically effective amount of an agonist for theangiotensin-(1-7) receptor in a pharmaceutically acceptable carrier,wherein a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist comprises an amount which is sufficient to inhibitcancer cell growth and/or proliferation.

[0049] Preferably, the cancer comprises cells having a functionalangiotensin-(1-7) receptor. The receptor may be located on the cellmembrane or intracellular. For example, the cancer may comprise cellswhose functional angiotensin-(1-7) receptor signals or secretes achemical or protein that inhibits cancer cell growth. Also, in anembodiment, the cancer is in a human subject. Also preferably, thecancer comprises bladder cancer, breast cancer, brain cancer, coloncancer, endometrial cancer, head and neck cancer, leukemia, lymphoma,lung cancer, melanoma, liver cancer, rectal cancer, ovarian cancer,prostate cancer, renal cancer, bone cancer, pancreatic cancer, or skincancer.

[0050] In an embodiment, the angiotensin-(1-7) receptor agonist of thecomposition comprises angiotensin-(1-7) peptide having the sequence setforth in SEQ ID NO: 1. In an embodiment, the angiotensin-(1-7) receptoragonist of the composition is modified to increase its chemicalstability in vivo. In an alternate embodiment, the angiotensin-(1-7)receptor agonist comprises a fragment of angiotensin-(1-7) or afunctional equivalent of angiotensin-(1-7) comprising conservative aminoacid substitutions, wherein conservative amino acid substitutions arethose substitutions which do not significantly effect the structure orfunction of the peptide. In yet another embodiment, theangiotensin-(1-7) receptor agonist comprises a non-peptide agonist.

[0051] Also preferably, the composition includes a compound whichincreases the efficacy or amount of circulating or cellularangiotensin-(1-7) agonist. In an embodiment, the compound whichincreases the efficacy or amount of angiotensin-(1-7) agonist increasesangiotensin-(1-7) synthesis. In another embodiment, the compound whichincreases the efficacy or amount of angiotensin-(1-7) agonist maydecrease angiotensin-(1-7) degradation, metabolism or clearance. In yetanother embodiment, the compound which increases the efficacy or amountof angiotensin-(1-7) agonist comprises a non-Ang-(1-7) angiotensinreceptor antagonist, such an antagonist of the AT₁ angiotensin receptor.

[0052] In an embodiment, a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist increases cellular prostacyclins. Inan embodiment, a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist increases cellular cAMP.

[0053] Also, in an embodiment, a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist increases the expression of genesinvolved in tumor suppression, apoptosis, and/or cell cycle inhibition.Preferably, the genes showing increased expression comprise BAD,oncostatin M-specific beta subunit, PDCD2, EGF response factor 1, CASP4,RBQ-3, p16-INK, menin, checkpoint suppressor 1, BAK, apoptotic proteaseactivating factor-1, SOCS-3, insulin-like growth factor binding protein2, B-myb or the fau tumor suppressor.

[0054] Alternatively, or additionally, a pharmaceutically effectiveamount of angiotensin- (1-7) receptor agonist may decrease the levels ofknown oncogenes, protein kinases, and/or cell cycle progression genes inthe cancer. Preferably, the genes showing decreased expression comprisecell cycle entry regulator, ERKI, cell cycle progression 2 protein,p21/K-ras 2B oncogene, epithelial cell kinase, ser/thr kinase, MAPkinase kinase 5 (MEK5), beta catenin, tyrosine-protein kinase receptortyro3 precursor, protein phosphatase 2A B56-alpha, cyclin-dependentkinase regulatory subunit (CDC28), cell division protein kinase 6(CDK6), c-myc oncogene, ERBB-3 receptor protein tyrosine kinase,A-kinase anchoring protein, or rho C.

[0055] In an embodiment, there is a discrete dosage range ofangiotensin-( 1-7) receptor agonist which is effective in inhibitingtumor cell growth. Preferably, the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-(l1-7) receptoragonist at the cancer which ranges from 0.005 nM to 10 μM. Morepreferably, the dose of angiotensin-(1-7) receptor agonist results in alocal concentration of angiotensin-(1-7) receptor agonist at the cancerwhich ranges from 0.05 nM to 1 μM. Even more preferably, the dose ofangiotensin-(1-7) receptor agonist results in a local concentration ofangiotensin-(1-7) receptor agonist at the cancer which ranges from 1 nMto 100 nM.

[0056] In another aspect, the present invention comprises a compositionto inhibit the growth of cancer cells in an individual comprising apharmaceutically effective amount of a compound which increases theefficacy or amount of circulating or cellular angiotensin-(1-7) agonistin a pharmaceutical carrier, wherein a pharmaceutically effective amountprovides endogenous levels of angiotensin-(1-7) receptor agonist whichis sufficient to inhibit cancer cell growth or proliferation. Forexample, in an embodiment, the method includes application of a compoundthat increases angiotensin-(1-7) synthesis. In another embodiment, thecompound which increases the efficacy or amount of cellularangiotensin-(1-7) agonist may decrease angiotensin-(1-7) agonistdegradation, metabolism or clearance. In yet another embodiment, thecompound which increases the efficacy or amount of cellularangiotensin-(1-7) agonist comprises a non- Ang-(1-7) angiotensinreceptor antagonist, such as an antagonist of the AT₁ angiotensinreceptor.

[0057] For example, such compounds may include ACE inhibitors, or anypharmaceutical that blocks either the AT₁ angiotensin II receptor. Suchcompounds act to cause an increase in Ang-(1-7) and thereby, cancontribute to Ang-(1-7) mediated inhibition of cancer growth. In anembodiment, the compounds comprise angiotensin receptor blockers.

[0058] Preferably, the cancer treated by the method of the presentinvention comprises bladder cancer, breast cancer, brain cancer, coloncancer, endometrial cancer, head and neck cancer, leukemia, lymphoma,lung cancer, melanoma, liver cancer, rectal cancer, ovarian cancer,prostate cancer, bone cancer, pancreatic cancer, skin cancer, or renalcancer.

[0059] In yet another aspect, the present invention comprises a kit forinhibiting cancer cell growth in an individual comprising: (a) at leastone container comprising a pharmaceutically effective amount of afunctional agonist for the angiotensin-(1-7) receptor, wherein apharmaceutically effective amount comprises an amount ofangiotensin-(1-7) receptor agonist which is sufficient to inhibit cancercell growth or proliferation; (b) a pharmaceutically acceptable carrier;and (c) instructions for use.

[0060] Preferably, the cancer cells comprise a functionalangiotensin-(1-7) receptor. The receptor may be located on the cellmembrane, or intracellular. In an embodiment, the cancer comprisesbladder cancer, breast cancer, brain cancer, colon cancer, endometrialcancer, head and neck cancer, leukemia, lymphoma, lung cancer, melanoma,liver cancer, rectal cancer, ovarian cancer, prostate cancer, bonecancer, pancreatic cancer, skin cancer, or renal cancer.

[0061] In an embodiment, the angiotensin-(1-7) receptor agonistcomprises angiotensin- (1-7) peptide having the sequence set forth inSEQ ID NO: 1. Preferably, the angiotensin- (1-7) receptor agonist usedin the kit is modified to increase its chemical stability in vivo. In anembodiment, the angiotensin-(1-7) receptor agonist comprises a fragmentof angiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions. Alternatively, theangiotensin-(1-7) receptor agonist, may comprise a non-peptide agonist.

[0062] Also preferably, the kit includes a compound which increases theefficacy or amount of cellular angiotensin-(1-7) agonist in the cells.In an embodiment, the compound which increases the efficacy or amount ofcellular angiotensin-(1-7) increases angiotensin-(1-7) synthesis. Inanother embodiment, the compound which increases the efficacy or amountof cellular angiotensin-(1-7) agonist may decrease angiotensin-(1-7)agonist degradation, metabolism or clearance. In yet another embodiment,the compound which increases the efficacy or amount of cellularangiotensin-(1-7) agonist comprises a non-Ang-(1-7) angiotensin receptorantagonist, such as an antagonist of the AT₁ angiotensin receptor.

[0063] Angiotensin-(i-7) is a Physiological Mediator of Cell Growth

[0064] Studies indicate that the angiotensin peptides may be associatedwith a variety of cellular activities. Still, angiotensin-(1-7) has longbeen considered an inactive product of Angll degradation. Only a fewstudies have implicated angiotensin peptides as potentially having arole in the regulation of cell growth and/or cancer. For example, in twostudies, patients receiving ACE inhibitors were found to have reducedrelative risk (0.73 and 0.79) of cancer (Jick, H. et al., Lancet, 1997,349:525-528; and Pahor, M. et al., Am. J. Hypertens., 1996, 9:695-699).These reductions in risk were not, however, statistically significant.In a retrospective study of 5207 patients in Scotland, the relativerisks of incident and fatal cancer among the 1559 patients treated withACE inhibitors were reduced, to 0.72 and 0.65, respectively, with therelative risk lowest in patients with lung or sex-specific cancer(Lever, A. F. et al., Lancet, 1998, 352:179-184). Other studiessuggested that treatment with ACE inhibitors may attenuate the growth ofpreneoplastic liver cells (Volpert, J. J. et al., J. Clin. Invest.,1996, 98:671-679) and renal cell carcinoma (Hii, S. I. et al., BritishJournal of Cancer, 1998, 77:880-883). While these studies suggest a rolefor ACE inhibitors in reducing cancer risk, there is no indication as tothe mechanism by which a lower risk of cancer may have occurred, or thatAng-(1-7) played a role.

[0065] The present invention describes the use of angiotensin-(1-7)[Ang-(1-7)] peptide and other Ang-(1-7) receptor agonists to inhibitcancer growth. Thus, in an embodiment, the present invention recognizesthat Ang-(1-7) peptide (Asp—Arg—Val—Tyr—Ile—His—Pro) (SEQ ID NO: 1)binds to a specific receptor present on tumor cells to affect secondmessengers associated with regulation of cell growth and proliferation.In an embodiment, the present invention describes that Ang-(1-7) bindsto specific receptors to invoke a decrease in the expression of genesassociated with cell growth and proliferation and to invoke an increasein expression of genes associated with suppression of cell proliferationand/or apoptosis and cell death.

[0066] Thus, in one embodiment, the present invention comprises a methodto inhibit cell proliferation comprising application ofangiotensin-(1-7) [Ang-(1-7)] or other agonists for theangiotensin-(1-7) receptor to the cells of interest. In anotherembodiment, the present invention comprises a method to inhibit cellproliferation comprising application of a compound which increases theefficacy or amount of cellular angiotensin-(1-7) to cells comprising theAng-(1-7) receptor. For example, and referring now to FIGS. 1-4,angiotensin-(1-7) inhibits vascular smooth muscle cell (VSMC) growthboth in vitro (FIGS. 1 and 2), and in vivo (FIGS. 3 and 4), suggestingthat Ang-(1-7) may act as an endogenous regulator of cell growth.

[0067] Thus, FIG. 1 shows the dose-dependent effect of angiotensinpeptides on 3H- thymidine incorporation into vascular smooth musclecells (VSMCs). It can be seen that Ang-(1-7) inhibits thymidineincorporation into DNA in a dose-dependent manner with an effectiveconcentration for 50% inhibition (IC50) of about 115 nM. FIG. 2 showsthat the ability of Ang-(1-7) to inhibit thymidine uptake is receptormediated. Thus, as shown in FIG. 2, the 1 μM Ang-(1-7)-mediatedreduction (+A7) in serum-stimulated growth (FBS) is inhibited by[Sar¹—Thr⁸]-Ang II (Sarthran) or [D—Ala⁷]-Ang-(1-7) (DalaA7) (which bindto the Ang-(1-7) receptor) but not by AT₁ (L158,809) or AT₂ (PD123177)receptor antagonists.

[0068]FIG. 3 shows stained sections of an uninjured rat carotid artery,a saline-treated injured carotid artery, and an injured corotid arterytreated with Ang-(1-7). Thus, in an embodiment, Ang-(1-7) reverses thecellular proliferation seen upon vascular injury. Morphometric analysisof carotid artery cross-sections indicates that Ang-(1-7) infusionsignificantly reduces the neointimal area compared to rats infused withsaline but has no effect on the medial area of the injured or thecontralateral uninjured artery as compared to saline controls (FIGS. 3and 4). Thus, Ang-(1-7) inhibits vascular growth in vivo and may preventvascular re-stenosis mediated by the proliferative response of smoothmuscle cells in blood vessels. For example, vascular re-stenosis is acomplication seen when vascular stents are used to prevent vesselocclusion in response to angioplasty and similar procedures.

[0069] The Effects of Ang-(1-7) Are Mediated by a Specific Receptor

[0070] In an embodiment, the effect of Ang-(1-7) on cell growth and/orproliferation is receptor mediated. Ang-(1-7) is a poor competitor atthe prototypical AT₁ angiotensin receptor in VSMC (Jaiswal, N. et al.,Hypertension, 1993, 21:900-905; and Jaiswal, N. et al., J. Pharmacol.Exp. Ther., 1993, 265:664-673) or the AT₂ angiotensin receptor(Chappell, M. C. et al., Peptides, 1995, 16:741-747; and Tallant, E. A.et al., Hypertension, 1991, 17:1135-1143). Thus, Ang-(1-7) displays IC₅₀levels in the micromolar range at the AT₁ or AT₂ angiotensin receptor(Tallant, E. A. et al., Hypertension, 1999, 34:950-957).

[0071] As described herein, IC₅₀ is the concentration of an agent whichprovides 50% of the total inhibition detected for a biological effect ofinterest, as for example, 50% inhibition of receptor binding or 50%inhibition of ³H-thymidine uptake.

[0072] Angiotensin receptors are pharmacologically defined by theirselectivity for the prototypical ligand losartan and similar antagonistssuch as L-1 58,809, while AT₂ receptors show selectivity for theantagonist PD 123177 or PD 123319 (de Gasparo et al., 1995). Ang II, bystimulation of AT₁ receptors, is a potent vasoconstrictor and stimulatesthirst and aldosterone release. Inhibition of its production or effectusing ACE inhibitors or AT₁ receptor antagonists reduces mean arterialpressure (Tallant, E. A. and Ferrario, C. M. Exp. Opin. Invest. Drugs1996, 5:1201-1214). In contrast, activation of AT₂ receptors by Ang IIis associated with vasodilation and reduced cell growth (Carey R. M. etal., Am. J. Hypertens. 2001, 6:98-1-2). [D—Ala⁷]-Ang-(1-7), a modifiedform of Ang-(1-7), selectively blocks responses to Ang-(1-7).[D—Ala⁷]-Ang-(1-7) is a poor competitor at the AT₁ or AT₂ receptor, anddoes not block pressor or contractile responses to Ang II (Britto, R. R.et al., Hypertension, 1997, 30:549-556; Fontes, M. A. P. et al., (BrainRes., 1994, 665:175-180; Oliveira, D. R. et al., Hypertension, 27:1998,1284-1290; and Santos, R. A. S. et al., Brain Res. Bull., 1994,35:293-298). Thus, an Ang-(1-7) binding site on bovine aorticendothelial cells [BAEC] which was competed for by [Sar¹—Ile⁸]-Ang TIand [D—Ala⁷]-Ang-(1-7) but not by losartan or PD123319 has beenidentified (Tallant, E. A. et al., Hypertension, 1996, 29:388-393; andHeitsch, H. et al., Hypertension, 2001, 37:72-76). A similar¹²⁵I-Ang-(1-7) binding site, sensitive to Ang-(1-7) and[D—Ala⁷]-Ang-(1-7), is found in the endothelium of canine coronaryartery rings (Ferrario, C. M. et al., Hypertension, 1997, 30:535-541),consistent with functional effects of Ang-(I-7) in canine and porcinecoronary arteries (Brosnihan, K. R. and Ferrario, C. M., Hypertension,1996, 27:523-528; and Porsti, I. et al., Br. J. Pharmacol., 1994,111:652-654). As described herein, a similar binding site for Ang-(1-7)has been identified on VSMCs (Iyer, S.N., et al., J. Cardiovasc.Pharmacol., 2000, 36:109-117).

[0073] Thus, there is a specific angiotensin-(1-7) [Ang-(1-7)] receptor,that is sensitive to [Sar¹—Thr⁸]-Ang II or [D—Ala⁷]-Ang-(1-7) but not tolosartan or PD123319. In an embodiment, the action of Ang-(1-7) toinhibit cell growth and/or cell proliferation comprises an interactionwith a specific receptor for Ang-(1-7). As described herein, thisangiotensin-(1-7) receptor may be referred to as the AT₍₁₋₇₎ receptor,in accordance with the guidelines established by the International Unionof Pharmacology Nomenclature Subcommittee for Angiotensin Receptors(Bumpus, F. M. et al., Hypertension, 1991, 17:720-721; and De Gasparo,M. et al., Hypertension, 1995, 25:924-927). The AT₍₁₋₇₎ receptor (orAng-(1-7) receptor) is defined by its sensitivity to Ang-(1-7), itsantagonism by [Sar¹—Thr⁸]-Ang II and [D—Ala⁷]-Ang-(1-7), and its lack ofresponse to losartan or PD123319, either functionally, or in competitionfor binding.

[0074] For example, and referring again to FIG. 2, inhibition ofmitogen-stimulated VSMC growth by Ang-(1-7) is not prevented by the AT₁antagonist L158,809 or the AT₂ antagonist PD 123319. However,[Sar¹—Thr⁸]-Ang II or the Ang-(1-7) antagonist [D—Ala⁷]-Ang-(1-7)effectively blocks growth inhibition of VSMCs by Ang-(1-7). Also, in anembodiment, the inhibition of serum-stimulated growth in cancer cells isattenuated by the selective Ang-(1-7) antagonist [D—Ala⁷]-Ang-(1-7), butnot by an AT₁ or AT₂ receptor antagonists (see FIG. 7, and discussionbelow).

[0075] In an embodiment, agonists other than Ang-(1-7) for the Ang-(1-7)receptor may be used in the methods of the present invention. In yetanother embodiment, non-peptide agonists such as those described in U.S.Pat. Nos. 6,429,222 and 6,235,766 (incorporated in their entireties byreference hererein) may be employed.

[0076] In an embodiment, the angiotensin-(1-7) or otherangiotensin-(1-7) receptor agonist is chemically modified to increaseits stability in vivo. For example, to increase stability, the peptidemay be modified at several positions to protect against aminopeptidaseand endopeptidase hydrolysis. For aminopeptidase protection, the amino(N) terminus of the peptide may be modified by substituting sarcosinefor aspartic acid (Asp) or acetylated aspartic acid for aspartic acid.To protect against endopeptidase attack, primarily ACE hydrolysis whichoccurs at the Ile⁵—His⁶ bond of Ang-(1-7), D-isoleucine and D-histidinemay be substituted for isoleucine at position 5 (Ile⁵) and histidine atposition 6 (His⁶), respectively, of the peptide. Additionally, a reducedor methyline isostere bond may be introduced between Ile⁵ and His⁶.

[0077] In yet another embodiment, the angiotensin-(1-7) orangiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(1-7) or a functional equivalent of angiotensin-(1-7) havingconservative amino acid substitutions, wherein conservative amino acidsubstitutions are defined to be those amino acid substitutions which donot affect the apparent structure, or inhibit the function, of thepeptide.

[0078] Angiotensin-(1-7) Inhibits Cancer Cell Growth and Proliferation

[0079] In an embodiment, the present invention describes the use ofagonists for the Ang- (1-7) receptor to inhibit growth and proliferationof cancer cells. Preferably, the Ang-(1-7) agonist may be used forinhibition of breast or lung cancer tumor growth (FIGS. 5-9). Theinhibition of tumor growth by Ang-(I-7) seen in vitro (FIGS. 5-8) isalso seen in vivo (FIG. 9) indicating that Ang-(1-7) is effective fortumor reduction in vivo.

[0080] Thus, Ang-(1-7) inhibits growth of human lung cancer cells(SK-LU-1, A549, SK-MES-1) and breast cancer cells (ZR-75-1), in adose-dependent manner (FIG. 5). In an embodiment, the dose of Ang-(1-7)required for inhibition of cancer cells comprises levels ofangiotensin-(1-7) used pharmacologically in animals or humans. Alsopreferably, the dose of angiotensin-(1-7) receptor agonist results in alocal concentration of angiotensin-(1-7) agonist at the tumor whichranges from 0.0005 nM to 10 ttM, and more preferably, from 0.05 nM to 1lM, or even more preferably, from 1 nM to 100 nM (FIG. 5).

[0081] Thus, as shown in FIG. 5, Ang-(1-7) reduced tumor cell growthwith an IC₅₀ of 0.05 nM for SK-LU-1 lung cancer cells, an IC₅₀ of 0.11nM for A549 lung cancer cells, an IC₅₀ of 0.4 nM for SK-MES-1 lungcancer cells, and an IC₅₀ of 0.02 nM for ZR-75-1 breast cancer cellsThese concentrations of Ang-(1-7) are well within the range of Ang-(1-7)doses used pharmacologically in animals or humans.

[0082] In an embodiment, the ability of Ang-(1-7) to inhibit tumorgrowth is a function of cell division and the length of the cell cycle.For example, the incorporation of ³H-thymidine into SK-LU-1, A549, andSK-MES-1 lung cancer cells and ZR-75-1 breast cancer cells stimulated togrow by the inclusion of 1% FBS is progressively reduced by dailyaddition of 100 nM Ang-(I-7) (FIG. 6). Thus, application of Ang-(1-7)may be hourly, daily, or over the course of weeks (FIG. 6).

[0083] Also, in an embodiment, the inhibition of serum-stimulated growthin cancer cells is attenuated by the selective Ang-(1-7) antagonist[D—Ala⁷]-Ang-(1-7), but not by AT₁ or AT₂ receptor antagonists (FIG. 7).Thus, inhibition of the serum-stimulated growth of SK-LU-1 human lungcancer cells by Ang-(1-7) is blocked by the Ang-(1-7) selectiveantagonist [D—Ala⁷]-Ang-(1-7), while neither ATI nor AT₂ angiotensinreceptor antagonists, Losartan and PD123177, respectively, are effective(FIG. 7). This suggests that the anti-proliferative effect of Ang-(1-7)in cancer cells is mediated by a novel AT₍₁₋₇₎ receptor.

[0084] Also, the effects of Ang-(1-7) on cell growth and proliferationare specific to Ang-(1-7), and are not exhibited by other angiotensinpeptides. Thus, neither Ang I, Ang-(2-8) or Ang III, Ang-(3-8) or AngIV, Ang-(3-7), nor Ang II mimicked the growth inhibitor effects ofAng-(1-7), as shown in FIG. 8. These results suggest that theanti-proliferative effect of Ang-(1-7) is mediated by a novel Ang-(1-7)receptor and may represent a new therapeutic treatment for thesecancers.

[0085] The effects of Ang-(1-7) on tumor growth are also seen in vivo.In a mouse model using athymic mice injected with breast cancer cells,tumor growth is dramatically reduced upon infusion of Ang-(1-7) (24μg/kg/hr) for 28 days. As shown in FIG. 9, an approximate 40% reductionin tumor volume is observed in mice treated with Ang-(1-7) for 4 weeks,while the tumor size doubles in the saline-treated animals, as comparedto tumor size prior to treatment. These results show that Ang-(1-7)inhibits breast tumor growth in vivo and that Ang-(1-7) is an effectivetherapeutic agent in vivo.

[0086] Angiotensin-(1-7) and Intracellular Signaling

[0087] One major response to treatment of cells, tissues or wholeanimals with Ang-(1-7) is the production of prostaglandins. Thus, in anembodiment, application of a pharmaceutically effective amount ofangiotensin-(1-7) or angiotensin-(1-7) receptor agonist increasesprostaglandins, prostacyclins and/or intracellular cAMP.

[0088] Ang-(1-7) induces prostaglandin release from astrocytes, porcineEC, and rat, porcine and rabbit VSMC (Jaiswal, N. et al., Hypertension,1992, 19:II-49-55; Jaiswal, N. et al., Am. J. Physiol. ReguL Integr.Comp. Physiol., 1991, 260:R1000-R1006; Jaiswal, N. et al., Hypertension,1993, 21:900-905; Jaiswal, N. et al., Hypertension, 1991, 17:1115-1120;Jaiswal, N. et al., J. Pharmacol. Exp. Ther., 1993, 264:664-673;Muthalif, M. M. et al., J. Pharmacol. Exp. Ther., 1998, 284:388-398; andTallant, E. A. et al., Hypertension, 1991, 18:32-39). For example, thevasodilator response to Ang-(1-7) and the depressor component of theresponse to Ang-(1-7) are reduced by prior treatment with thecyclooxygenase inhibitor indomethacin, indicating that these responseswere mediated by prostaglandins (Benter, I. F. et al., Peptides, 1993,14, 679-684; Meng, W. and Busija, D. W., Stroke, 1993, 24:2041-2045; andIyer, S. N. et al., J. Cardiovasc. Pharmacol., 2000, 36:109-117).

[0089] Prostacyclin (PGI₂) is a type of prostaglandin. Prostacylin is apotent vasodilator and reduces vascular growth via production of cAMP.Prostacyclin is produced by the cyclooxygenase-mediated conversion ofarachidonic acid into PGG₂/PGH₂, which is subsequently processed byprostacyclin synthase into prostacyclin. Interestingly, thecyclooxygenase inhibitor indomethacin effectively blocks the growthinhibition mediated by Ang-(1-7).

[0090] The addition of prostacyclin (or stable analogs of prostacyclinsuch as carbacyclin) to VSMCs activates adenylate cyclase resulting inan elevation in the cellular levels of cAMP. Ang-(1-7), at aconcentration of 1 μM, causes a significant increase in the cellularlevels of cAMP, to 131.9±9.7% of basal (n=3, p<0.05), in the presence of1 mM isobutylmethyl xanthine (IBMX), a cyclic nucleotidephosphodiesterase inhibitor. cAMP activates a cAMP-dependent proteinkinase, protein kinase A. As shown in FIG. 10, the reduction inserum-stimulated ³H-thymidine incorporation by Ang-(1-7) or carbacyclinwas completely blocked by pretreatment with the protein kinase Ainhibitor (PKAI) Rp-adenosine-3′,5′-cyclic monphospho- phorothioatetriethylamine salt (Rp-cAMPS). These results suggest that Ang-(1-7) isdirectly coupled to the Gs protein to activate adenylate cyclase andelevate cellular cAMP production. Alternatively, Ang-(1-7) may stimulatethe production of prostacyclin which binds to prostacyclin receptorscoupled to adenylate cyclase and the synthesis of cAMP. Thus, in anembodiment, Ang-(1-7) causes an increase in the cellular levels of cAMP(directly or via prostacylin) which stimulates the cAMP-dependentprotein kinase to inhibit growth.

[0091] Alternatively and/or additionally, Ang-(1-7) may inhibit cellgrowth by preventing the phosphorylation and activation of MAP kinasesin response to mitogen stimulation. Compounds that increase theintracellular concentration of cAMP have been shown to reduce MAP kinaseactivity in VSMCs and fibroblasts and thereby inhibit mitogen-stimulatedgrowth in VSMCs (Cook, S. J. and McCormick, F., Science, 1993,262:1069-1072; and Wu, J. et al., Science, 1993, 262;1065-1068). Inaddition, classic growth factors, such as PDGF, epidermal growth factor,and basic fibroblast growth factor stimulate VSMC growth in vitro and invivo. Growth stimulation by these mitogens as well as by Ang II ismediated, at least in part, through activation of MAP kinases to induceearly response genes and increase transcription.

[0092] The activity of the MAP kinases ERKI and ERK2 in VSMCs can bemeasured using phospho-specific antibodies that only recognize theactivated protein kinases. As shown in FIG. 11, Ang-(1-7) causes adose-dependent reduction in Ang II-stimulated ERK activity in VSMCs,with maximal inhibition at 1 IM Ang-(1-7). Similarly, Ang-(1-7) at 1 μMshows maximal inhibition of serum-stimulated ERKl and ERK2 activation inSK-LU-1 lung cancer cells (FIG. 12).

[0093] Ang-(1-7) also reduces ERK phosphorylation by platelet derivedgrowth factor (PDGF). Thus, 10 ng/mL PDGF increases ERKI and ERK2activities by 16-fold and 26-fold in VSMCs. This stimulation isinhibited by almost 50% by 1 JIM Ang-(1-7). Also, as shown in FIG. 13,Ang-(1-7) inhibits platelet-derived growth factor (PDGF) or epidermalgrowth factor (EGF)-stimulated ³H-thymidine incorporation into humanZR-75-1 breast cancer cells by at least 50% (FIG. 13).

[0094] Thus, in an embodiment, Ang-(1-7) inhibits cell growth through areduction in the activity of mitogen-stimulated MAP kinases. Ang-(I-7)may also reduce MAP kinase activity by inhibiting the signaling pathwaysthat stimulate MAP kinase phosphorylation or by stimulating MAP kinasephosphatase activity.

[0095] Signal Transduction and Potential Molecular Mechanisms ofInhibition of Cancer Cell Growth and Proliferation by Ang-(1-7)

[0096] Cell division is a complex process that occurs with exquisiteprecision such that each daughter cell receives the correct number ofchromosomes and is capable of independent function. Cell cycle eventsare initiated at the appropriate time, allowing for the completion ofone phase before the next one is triggered. In an embodiment,angiotensin-(1-7) or other angiotensin-(1-7) receptor agonists inhibitcancer cell growth and/or proliferation by increasing the expression ofgenes involved in tumor suppression, apoptosis, and/or cell cycleinhibition. Alternatively, or additionally, angiotensin-(1-7) or otherangiotensin-(1-7) receptor agonists may inhibit cancer cell growthand/or proliferation by decreasing the levels of known oncogenes,protein kinases, and/or cell cycle progression genes in the cancer. FIG.14 shows a histogram of gene transcription increases and decreases whenquiescent SK-LU-1 lung cancer cells stimulated with 1% FBS for 2 h inthe presence of 100 nM Ang-(1-7) as compared to SK-LU-1 lung cancercells stimulated with 1% FBS for 2 h in the absence of Ang-(1-7).

[0097] Cyclin-dependent kinases (CDKs) are proteins involved in thecontrol of the cell cycle. CDKs catalyze the covalent attachment ofphosphate to protein substrates, thereby altering the enzyme activity orprotein affinity of the substrate. The regulation of the cellularconcentrations of CDKs leads to cyclical changes in the phosphorylationof key components of the cell-cycle machinery, resulting in theinitiation (or inhibition) of cell cycle events. CDKs are activated bybinding to regulatory proteins called cyclins. Changes in CDK activityduring the cell cycle are due primarily to the amount of cyclin proteinsin the cell. In turn, cyclin concentrations are regulated attranscription and through proteolytic degradation of the cyclins atspecific cell-cycle stages.

[0098] For example, two classes of CDK inhibitors—the p16/p19^(ARF) andp21 families of proteins—bind to specific CDKs to prevent theirinteraction with cyclins and thereby interfere with cyclin/CDKregulation of the cell cycle. Thus, p16^(INK4a) inhibits CDK4 and CDK6,resulting in hypophosphorylation of the retinoblastoma protein (Rb).When Rb is under-phosphorylated, it binds to the transcriptionalactivator E2F to block transcription and prevent progression through thecell cycle. Upon phosphorylation by CDK4 and 6, hyperphosphorylated Rbreleases bound E2F, allowing it to increase the transcription of genesinvolved in progression through the cell cycle (Lukas, J. et al.,Nature, 1995, 375:503-506; and Serraro, M. et al., Science, 1995,267:249-252).

[0099] Cell cycle control of most cell types is also responsive toextracellular signals. Classic growth factors such as platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF), basic fibroblastgrowth factor (bFGF), or serum stimulate cell growth in vitro and invivo. Growth stimulation by these mitogens is mediated, at least inpart, through ras-Raf activation of mitogen-activated protein kinases(MAP kinases) to induce early response genes and increase transcription(Marrero, M. B. et al., J. Biol. Chem., 1997, 272:24684-24690; Molloy,C. J. et al., J. Biol. Chem, 1993, 268:7338-7345; and Pelech, S. L. andSanghera, J. S., Science, 1992, 257:1355-1356).

[0100] The Ras/Raf/MEK/ERK phosphorylation cascade is the prototypicalcellular proliferation pathway documented in mammalian cells. Growthstimulation by many cytokines as well as by growth hormones alsoinvolves activation of the Janus kinase (JAK) family of cytosolictyrosine kinases. Janus kinases stimulate the phosphorylation of STAT(signal transducers and activators of transcription) proteins, causingtheir translocation to the nucleus and subsequent activation oftranscription (Marrero, M. B. et al., J. Biol. Chem., 1997,272:24684-24690; and Marrero, M. B. et al., Nature, 1995, 375:247-250).

[0101] Thus, in an embodiment, application of a pharmaceuticallyeffective amount of angiotensin-(1-7), or other angiotensin-(1-7)receptor agonists may inhibit cancer cell growth and/or proliferation bydecreasing the levels of known oncogenes, protein kinases, and/or cellcycle progression genes in cancer cells. In an embodiment, the genesshowing decreased expression comprise cell cycle entry regulator, ERK1,cell cycle progression 2 protein, p21/K-ras 2B oncogene, epithelial cellkinase, ser/thr kinase, MAP kinase kinase 5 (MEK5), beta catenin,tyrosine-protein kinase receptor tyro3 precursor (FIG. 14). As shown inFIG. 15, Ang-(1-7) decreases both MEK 5 mRNA and protein levels inSK-LU-l lung cancer cells stimulated with serum. Thus, although MEK5protein levels increased immediately following mitogen stimulation, atboth 4 and 8 hours MEK5 mRNA and protein levels are reduced by treatmentwith Ang-(1-7).

[0102] Thus, in an embodiment, Ang-(1-7) agonists inhibit cancer cellgrowth by regulation of MAP kinase and JAK/STAT signaling pathways. Thisis supported by the findings that: (1) p21/ras mRNA and ERKI mRNA andprotein are downregulated in serum-stimulated human SK-LU-1 lung cancercells following Ang-(1-7) treatment; (2) down-regulation of MEK5 mRNAand protein is observed in mitogen-stimulated human lung cells followingtreatment with Ang-(1-7); (3) Ang-(1-7) up-regulates the expression ofSOCS-3, a negative regulator of the JAK/STAT pathway in human lungcancer cells (FIG. 14) (Dey, B. R. et al., Biochem. Biophys. Res.Commun., 2000, 278:38-43; and Duhe, R. J. et al., Cell Biochem.Biophys., 2001, 34:17-59); and (4) Ang-(1-7) inhibits tumor growth invivo.

[0103] Alternatively and/or additionally, Ang-(1-7) agonists inhibitcancer cell growth and/or proliferation by increasing the levels ofgenes involved in tumor suppression and/or cell cycle inhibition.Preferably, the genes showing increased expression comprise pl6-INK,oncostatin M-specific beta subunit, PDCD2, EGF response factor 1, CASP4,RBQ-3, menin, checkpoint suppressor 1, SOCS-3, insulin-like growthfactor binding protein 2, B-myb or the fau tumor suppressor (FIG. 14).

[0104] Thus, under normal conditions, tissues maintain a balance betweenthe rates of cell proliferation and cell death. In contrast, tumorformation is a pathological state resulting from heightened celldivision and a reduced rate of apoptosis. Many cancer cells manifest anenhanced resistance to physiological stimuli that would ordinarilytrigger apoptosis in normal cells. Substances that can stimulateapoptosis in cancer cells may provide a novel mechanism for reducingcell number.

[0105] Caspase-3 is activated during apoptosis. In an embodiment,treatment of cancer cells with Ang-(1-7) upregulates genes encoding thepro-apoptotic proteins BAD, BAK as well as apoptotic protease activatingfactor 1 (FIG. 14) and increases the caspase-3 cleavage product ofpoly(ADP-ribose) polymerase (PARP) (FIG. 16) in mitogen-stimulatedcancer cells. An increase in the amount of caspase-3 cleavage productPARP by treatment with Ang-(1-7) indicates that Ang-(1-7) stimulatesapoptosis in lung cancer cells to thereby reduce cell growth.

[0106] Therapeutics

[0107] The invention contemplates methods of administration which arewell known in the art. For example, in an embodiment, administration ofthe compound is intravenous. In another embodiment, the method ofadministration is by a transdermal patch. Also, administration mayemploy a time-release capsule. In another embodiment, administration ofthe compound is intra-arterial. In yet another embodiment,administration of the compound is oral or as an aerosol. In anotherembodiment, administration of the compound is sublingual. In yet anotherembodiment, administration of the drug is transrectal, as by asuppository or the like.

[0108] Pharmaceutical formulations can be prepared by procedures knownin the art. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers, that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivates; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agentssuch as agar, calcium carbonate, and sodium bicarbonate; agents forretarding dissolution such as paraffin; resorption accelerators such asquaternary ammonium compounds; surface active agents such as cetylalcohol, glycerol monostearate; adsorptive carriers such as kaolin andbentonite; and lubricants such as talc, calcium and magnesium stearate,and solid polyethyl glycols.

[0109] The compounds can also be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

[0110] The therapeutic efficacy of exogenous compounds can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals using procedures known in the art. The dose ratio between toxicand therapeutic effects is the therapeutic index and may be expressed asLD₅₀/ED₅₀, wherein LD₅₀ is understood to represent the dose which istoxic to 50% of the subjects and ED₅₀ is understood to represent thedose which is effective in 50% of the subjects. Generally, compoundswhich exhibit large therapeutic indices are preferred. Administration ofthe compound may be hourly, daily, weekly, monthly, yearly or a singleevent.

[0111] In an embodiment, the dose of Ang-(1-7) agonist required forinhibition of cancer cells comprises levels of angiotensin-(1-7) agonistthat are used pharmacologically in animals and humans. Also preferably,the dose of angiotensin-(1-7) receptor agonist results in a localconcentration of angiotensin-(1-7) agonist at the tumor which rangesfrom 0.005 nM to 10 μM, and more preferably, from 0.05 nM to 1 μM, oreven more preferably, from 1 nM to 100 nM. Also, the ability ofAng-(1-7) agonists to inhibit tumor growth may a function of celldivision and the length of the cell cycle. Thus, application of theAng-(1-7) agonist may be hourly, daily, or over the course of weeks.Thus, preferably, the effective amount of the Ang-(1-7) agonistcomprises from about 1 ng/kg body weight to about 100 mg/kg body weight.More preferably, the effective amount of the Ang-(1-7) agonist comprisesfrom about 1 μg/kg body weight to about 50 mg/kg body weight. Even morepreferably, the effective amount of the Ang-(1-7) agonist compoundcomprises from about 10 μg/kg body weight to about 10 mg/kg body weight.Alternatively, a continuous level of Ang-(1-7) agonist ranging fromabout 0.05-1,000 μg/kg/hour, or more preferably, 0.5-250 μg/kg/hr, oreven more preferably 5-50 μg/kg/hour may be employed. The actualeffective amount will be established by dose/response assays usingmethods standard in the art. Thus, as is known to those in the art, theeffective amount will depend on bioavailability, bioactivity, andbiodegradability of the compound.

EXAMPLES

[0112] The present invention will be further understood by reference tothe following non-limiting examples.

Example 1

[0113] Materials and Methods

[0114] A. Angiotensin Receptor Pentides and Non-Peptide Compounds

[0115] All angiotensin peptides (natural and modified) were obtainedfrom Bachem, Torrance, Calif. AT₁ antagonists Losartan and LI 58,809were obtained from Merck & Co., Inc., Rahway, N.J. The AT₂ antagonistPD123177 was obtained from Parke-Davis Pharmaceutical Research, AnnArbor, Mich.

[0116] B. Rat Vascular Smooth Muscle Cells (VSMCs) and Human Lung CancerCells

[0117] Vascular smooth muscle cells were isolated form 12-14 week oldSprague-Dawley rats by explant culture (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108). Human lung and breast cancer cell lineswere obtained from American Type Tissue Culture (ATTC) and includedcells of the SK-LU-1 and A549 cell lines (both of which are derived fromadenocarcinomas) as well as SK-MES-1 cells (derived from non-small celllung tumors) and ZR-75-1 breast cancer cells.

[0118] Cells were grown in DMEM with 10% fetal bovine serum (FBS), 100μg/mL penicillin and 100 units/mL streptomycin in a humidified 37° C.incubator gassed with 5% CO₂ and 95% room air. Cells were grown tosubconfluence in either 24-well cluster plates or 100 mm dishes and madequiescent by treatment for 48 h with serum-depleted growth media, priorto the experiments outlined below to measure cell growth (³H-thymidineincorporation), cell signaling or apoptosis.

[0119] C. Analysis of ³H-Thymidine Incorporation

[0120] To measure ³H-thymidine incorporation, quiescent cells wereincubated with serum and angiotensin peptides for 24 h at 37° C.³H-thymidine (0.25 μCi/well) was added and the cells incubated for anadditional 4 h to incorporate the radiolabeled nucleotide. Subsequently,the cell monolayer was washed with cold phosphate-buffered saline (PBS;50 mM NaPO₄, 120 mM NaCl, pH=7.2). The adherent cells were precipitatedwith cold 10% TCA (4° C. for 30 min) and dissolved in 0.2% SDS in 0.1 NNaOH. Incorporated ³H-thymidine was determined by liquid scintillationspectrometry, as previously described (Freeman, E. J. et al.,Hypertension, 1996, 28:104-108). Growth inhibition was defined as areduction in the amount of ³H-thymidine incorporation as compared to themitogen-stimulated controls.

[0121] To study the receptor specificity of the effect, cell monolayerswere preincubated with 1 μM of the AT₁ antagonist Losartan (orL158,809), the AT₂ antagonist PD 123319, the non-selective angiotensinpeptide antagonist [Sar¹—Thr⁸]-Ang II, or the Ang-(1-7)-selectiveantagonist [D—Ala⁷]-Ang-(1-7), followed by treatment with various dosesof mitogens and Ang-(1-7). Quiescent cells were stimulated withincreasing concentrations of Ang-(1-7) and/or antagonists for 24 h.During an additional 4 h, cell monolayers were pulsed with ³H-thymidine(0.25 μCi/well) and harvested. Cells treated with mitogen andantagonists in the absence of Ang-(1-7) were used as the controls, todetect any effect of the antagonists alone.

[0122] D. Statistics

[0123] For all experiments, cells were used from at least threedifferent passage numbers of each cell type. Values are expressed asmean±standard error of the mean. Statistical significance of differenceswas evaluated by one way analysis of variance with p values corrected byDunnett's post test, using the statistics package Instat (GraphPad). Thecriterion for statistical significance was set at p<0.05.

[0124] E. RT-PCR and Western Analysis

[0125] For RT-PCR, total RNA was isolated using the Atlas Pure Total RNALabeling System (Clontech Laboratories, Inc). The RNA concentration wasquantified by UV spectroscopy and any degradation assessed by ethidiumbromide staining intensity of 28S and 18S ribosomal RNA followingagarose gel electrophoresis. The isolated RNA was incubated with DNaseto eliminate any residual DNA, and approximately 250 ng of total RNA persample incubated with or without AMV reverse transcriptase in a mixturecontaining deoxynucleotides, random hexamers, and RNase inhibitor inreverse transcriptase buffer. The mixture was heated for 5 min at 95° C.to terminate the reaction. For amplification of the resulting cDNA, 1μmol/L gene-specific primers, 0.2 mmol/L deoxynucleotides, 5μCi³²P-dCTP, 1.5 mmol/L MgCl₂, and 1.5 U Taq DNA polymerase was added to3 μL of the RNA sample in a final volume of 50 μL. As an internalstandard, primers specific for the gene encoding Elongation Factor 1αwere added. Following PCR, the amplification products were separated bypolyacrylamide gel electrophoresis, visualized by autoradiography, andanalyzed using the MCID imaging system.

[0126] For Western blot analysis, quiescent cells treated with Ang-(1-7)and serum for various periods of time, between 2 and 24 h, weresolubilized in SDS and protein content analyzed using the modified Lowrymethod (Lowry, O. H., et al., J. Biol. Chem., 1951, 193:265-275).Proteins were separated electrophoretically on SDS polyacrylamide gels,transferred to polyvinyl membranes, and incubated with primaryantibodies to proteins of interest. Appropriate horseradish peroxidase(HRP)-conjugated second antibodies were added and immunoreactiveproducts visualized using the enhanced chemoluminescence reagents fromAmersham. The density of each immunoreactive product was quantifiedusing the MCID imaging system. Antibodies to proteins that participatein cell signaling, apopotosis, and regulation of the cell cycle arecommercially available from a variety of sources.

[0127] F. Measurement of MAP Kinases

[0128] Quiescent lung cancer cells were incubated with increasingconcentrations of Ang-(1-7) [from 10⁻⁹ to 10⁻⁶ M] for 10 min at roomtemperature. Reactions were terminated with Triton lysis buffer (50 mMTris-HCI, pH 7.4, 1% Triton X-100, 100 mM NaCl, 5 mM EDTA, 50 mM NaF,0.6tM leupeptin, 0.01 mM Na₃VO₄ and 0.1 mM PMSF) and proteinconcentrations determined (Lowry, O.H., et al., J. Biol. Chem., 1951,193:265-275). Proteins were separated by SDS polyacrylamide gelelectrophoresis and transferred to polyvinyl membranes. The activationand autophosphorylation of ERKI and ERK2 was determined using antibodiesspecific for the phosphorylated kinases (using antibodies from CellSignaling Technologies). The immunoreactive product were visualized byenhanced chemiluminescence (ECL, Amersham) and quantified bydensitometry, using the MCID image analysis system. The phospho-MAPkinase antibodies only recognize the catalytically activated andphosphorylated forms of MAP kinase (both ERK1 and ERK2 MAP kinase). Theblots were also probed with antibodies to ERK1/2, to control for proteinloading.

Example 2

[0129] Inhibition of Vascular Growth by Ang-(1-7) In Vitro

[0130] These experiments support earlier indications (Freeman, E. J. etal., Hypertension, 1996, 28:104-108; and Tallant, E. A. et al.,Hypertension, 1999, 34:950-957) that Ang-(1-7) inhibits the growth ofcultured vascular smooth muscle cells (VSMCs). Incorporation of³H-thymidine into VSMCs obtained from rat thoracic aorta wassignificantly increased by incubation with fetal bovine serum (FBS),platelet-derived growth factor (PDGF), or Ang II. Following a 48 hrtreatment with 1 μM Ang-(1-7), the incorporation of ³H-thymidine inresponse to 1% FBS, 10 ng/mL PDGF and 100 nM Ang II was markedlyattenuated (to 66.4, 84.3, and 75.8% of mitogen-stimulated activity,respectively). The reduction in serum-stimulated thymidine incorporationby Ang-(1-7) was dose-dependent, with a peak effect at a dose of 1 tMand an IC₅₀ of 115 nM (FIG. 1). Maximal inhibition by 1 lM Ang-(1-7) wasapproximately 60% of control in the presence or absence of FBS, which issimilar to the growth inhibition previously reported for atrialnatriuretic factor (ANF) (Appel, R. G., Am. J. Physiol., 1990,259:E312-E318).

[0131] Total cell number in response to treatment with Ang-(1-7) wasalso determined using a Coulter counter. The number of cells per wellincreased to 142% of basal following treatment with 1% serum. Treatmentof serum-stimulated cells with 1 JIM Ang-(1-7) significantly reduced thenumber of cells per well (to 109% of basal). By comparison, Ang IIincreased the number of cells per well to 145% of basal values andcaused a dose-dependent stimulation of ³H-thymidine incorporation intoVSMCs, as shown in FIG. 1. Thus, Ang-(1-7) inhibits mitogen-stimulatedVSMC growth and opposes the proliferative effects of Ang II.

Example 3

[0132] The Effects of Ang-(1-7) Are Mediated Via a Specific Ang-(1-7)Receptor

[0133] It is documented that the mitogenic effect of Ang II is mediatedby the AT₁ angiotensin receptor and that stimulation of vascular AT₂receptors inhibits growth. However, attenuation of (fetal bovine)serum-stimulated thymidine incorporation (FBS) by Ang-(1-7) (+A7) wasunaffected by antagonists selective for AT₁ (L1 58,809) or AT₂ (PD123177) receptors (FIG. 2). In contrast, a 10-fold molar excess of thenon-selective angiotensin receptor antagonist ([Sar¹—Thr⁸]-Ang II;Sarthran) completely blocked growth inhibition by Ang-(1-7), indicatingthat the effect of the heptapeptide was a result of the activation of anangiotensin receptor pharmacologically distinct from either AT₁ or AT₂receptors. [D—Ala⁷]-Ang-(1-7) also blocked the growth inhibitoryresponse to Ang-(1-7). The substitution of D-alanine for proline inAng-(1-7) results in a molecule that has no agonistic activity, does notcompete at AT₁ or AT₂ receptors, and selectively blocks hemodynamic andrenal responses to Ang-(1-7) (Santos, R. A. S. et al., Brain Res. Bull.,1994, 35:293-298). These data indicate that Ang-(1-7) inhibits VSMCgrowth through activation of a non-AT₁, non-AT₂ receptor that issensitive to [Sar¹—Thr⁸]-Ang II and [D—Ala⁷] -Ang-(1-7), the AT₍₁₋₇₎receptor.

Example 4

[0134] In Vivo Studies of Ang-(1-7) Reduction of Vascular Cell Growth

[0135] To study the role of the peptide in vivo, the effect of Ang-(1-7)on vascular growth stimulated by balloon catheter injury to the ratcarotid artery was determined. Intravenous infusion of Ang-(1-7) with achronically implanted minipump [24 μg/kg/h, 5 liL/h, 12 days] increasedthe plasma Ang-(1-7) concentration to 131.4±39.7 pM (n=5) from 42.2±10.7pM (n=8) in carotid artery-injured rats infused with saline. Plasmaconcentrations of Ang II, blood pressure and heart rate were similar inrats infused with Ang-(1-7) or saline. Morphometric analysis of carotidartery cross-sections indicated that Ang-(1-7) infusion significantlyreduced the neointimal area compared to rats infused with saline(0.10±0.009 mm² vs. 0.066±0.012 mm², respectively; p<0.05) but had noeffect on the medial area of the injured or the contralateral uninjuredartery as compared to saline controls (FIGS. 3 and 4) (Strawn, W. B. etal., Hypertension, 1999, 33:207-211; and Tallant, E. A. et al.,Hypertension, 1999, 34:950-957). Thus, Ang-(1-7) inhibits vasculargrowth in vivo. The antiproliferative effect of Ang-(1-7) in preventingneointimal growth is of clinical importance as vascular re-stenosismediated by a proliferative response of smooth muscle in blood vesselsis a complication of surgical procedures that use stents to preventvessel occlusion following crushing of an atherosclerotic plaque(angioplasty).

[0136] The concentrations of Ang-(1-7) shown to be effective in reducingvascular growth in response to injury are similar to plasma levels ofAng-(1-7) in rats following balloon catheter injury and treated with theACE inhibitor lisinopril (20 mg/kg/day for 14 days). Inlisinopril-treated rats, plasma levels of Ang-(1-7) were elevated2.3-fold (from 42.3±6.7 pM in saline treated animals (n=10) to 99.1±6.7pM (n=10)). In these same animals, the cross-sectional area of theneointima was reduced to 0.09±0.01 mm² as compared to 0.12±0.02 mm² insaline-treated controls (p<0.05). Thus, exogenous Ang-(1-7) infusion ortreatment with the ACE inhibitor lisinopril to increase Ang-(1-7)reduced neointimal formation after vascular injury at concentrations ofthe peptide only two-fold higher than in saline-treated rats.

Example 5

[0137] Inhibition of Human Cancer Cell Growth by Ang-(1-7)

[0138] These experiments show that Ang-(1-7) reduces the growth of lungand breast cancer cells. Ang-(1-7) inhibited serum-stimulated³H-thymidine incorporation into human lung cancer cells of the A549,SK-MES-1, and SK-LU-1 cell lines and the ZR-75-1 breast cancer cellline. The attenuation of human lung adenocarcinoma SK-LU-1 cell growthwas dependent on the dose of Ang-(1-7) with a maximal reduction of33.8±5.3% of serum-stimulated growth and an IC₅₀ of 0.05 nM, as shown inFIG. 5. Ang-(1-7) also attenuated mitogen-stimulated growth of humanlung adenocarcinoma A549 cells (maximal inhibition of 41.3±10.9%,IC₅₀=0.11 nM) as well as non-small cell lung cancer SK-MES-1 cells(maximal inhibition of 40.9±2.9%, IC₅₀=0.4 nM) and breast cancer ZR-75-1cells (maximal inhibition of 37.2±6.1; IC₅₀=0.02 nM). Thus, Ang-(1-7)reduces human lung and breast cancer cell growth in a dose-dependentmanner with IC₅₀ levels similar to circulating levels of Ang-(1-7)measured after treatment of rats with the ACE inhibitor lisinopril(Campbell, D. J. et al., Hypertension, 1993, 22:513-522; and Kohara, K.et al., Circulation, 1991, 84 (supp. II):662).

[0139] The inhibition of growth by Ang-(1-7) was also dependent upon thetime of treatment with Ang-(1-7). The incorporation of ³H-thymidine intoSK-LU-1, A549, and SK-MES-1 lung cancer cells and ZR-75-1 breast cancercells stimulated to grow by the inclusion of 1% FBS was progressivelyreduced by daily addition of 100 nM Ang-(1-7), as shown in FIG. 6.Ang-(1-7) was renewed daily due to the endogenous degradation of thepeptide (Chappell, M. C. et al., Hypertension, 1998, 31:362-367). Theseresults suggest that Ang-(1-7), an endogenous peptide, inhibits themitogen-stimulated growth of lung cancer cells.

[0140] Inhibition of the serum-stimulated growth of SK-LU-1 human lungcancer cells by Ang-(1-7) was blocked by the Ang-(1-7) selectiveantagonist [D—Ala⁷]-Ang-(1-7), while neither AT₁ nor AT₂ angiotensinreceptor antagonists Losartan and PD123177, respectively, were effective(FIG. 7). This suggests that the anti-proliferative effect of Ang-(1-7)in lung cancer cells is mediated by a novel AT₍₁₋₇₎ receptor.

[0141] Also the effects are specific to Ang-(1-7), and are not exhibitedby other angiotensin peptides. Thus, neither Ang I, Ang-(2-8) or AngIII, Ang-(3-8) or Ang IV, Ang-(3-7), nor Ang II mimicked the growthinhibitor effects of Ang-(1-7), as shown in FIG. 8. These resultssuggest that the anti-proliferative effect of Ang-(1-7) is mediated by anovel Ang-(1-7) receptor and may represent a new therapeutic treatmentfor these cancers.

Example 6

[0142] Inhibition of Tumor Growth by Ang-(1-7)

[0143] To determine whether Ang-(1-7) inhibits tumor growth in vivo,athymic mice were inoculated subcutaneously in the lower flank withapproximately 1.5×10⁷ cells of the ZR-75-1 breast cancer cell line.Tumor volumes were measured by caliper two times per week and calculatedusing the formula for a semiellipsoid. After 40 days, the mice hadtumors approximately 175 mm³ in size and were randomized for treatment.Primed osmotic mini-pumps (delivery rate of 0.25 μL/hr) were implantedonto the backs of the mice with the control group receiving continuousintravenous infusion of saline (6 μL/24 hr) and the experimental groupreceiving Ang-(1-7) (24 μg/kg/hr) for 28 days. The dose of Ang-(1-7) wasbased on previous studies with rats, which indicated that this dose wastolerated with no change in weight, blood pressure, or heart rate andresulted in a 2 to 3-fold elevation in circulating Ang-(1-7) (Strawn, W.B. et al., Hypertension, 1999, 33:207-211. As shown in FIG. 9, anapproximate 40% reduction in tumor volume was observed in mice treatedwith Ang-(1-7) for 4 weeks, while the tumor size doubled in the saline-treated animals, as compared to tumor size prior to treatment. Theseresults show that Ang-(1-7) inhibits breast tumor growth in vivo andthat Ang-(1-7) is an effective therapeutic agent in vivo.

Example 7

[0144] Mechanism of Growth Inhibition by Ang-(1-7).

[0145] It was found that Ang-(1-7) stimulates prostacyclin (PGI₂)release from VSMCs isolated from Sprague-Dawley rat aortas, measured asthe release of the stable metabolite of prostacyclin, 6-keto-PGF_(1α).Ang-(1-7) caused a dose-dependent release of prostacyclin, with maximalrelease of 177.9±25.2% above basal release at 100 nM Ang-(1-7). Sinceprostacyclin inhibits VSMC growth, these results suggest that Ang-(1-7)attenuates vascular growth through the production and release ofprostacyclin.

[0146] Prostacyclin is produced by the cyclooxygenase-mediatedconversion of arachidonic acid into PGG₂/PGH₂, which is subsequentlyprocessed by prostacyclin synthase into prostacyclin. Interestingly, thecyclooxygenase inhibitor indomethacin (IND, 10 μM) effectively blockedthe growth inhibition mediated by Ang-(1-7) (97.4±3.6% of control, n=4,p<0.05) compared to the decrease of serum-stimulated ³H-thymidineincorporation by Ang-(1-7) in the absence of indomethacin (79.1±5.1% oftotal, n=5, p<0.05). Since neither a lipoxygenase inhibitor nor thecytochrome P450 inhibitor 1 7-octadecynoic acid had any effect on growthinhibition by the heptapeptide, these results show that Ang-(1-7)inhibits VSMC growth through the production of a metabolite of thecyclooxygenase pathway which may be prostacyclin.

[0147] The addition of prostacyclin or carbacyclin (5 μM; Calbiochem, LaJolla Calif.) (a stable analogs of prostacyclin) to VSMCs activatesadenylate cyclase resulting in an elevation in the cellular levels ofcAMP. Ang-(1-7), at a concentration of 1 μM, caused a significantincrease in the cellular levels of cAMP, to 131.9±9.7% of basal (n=3,p<0.05), in the presence of 1 mM isobutylmethyl xanthine (IBMX), acyclic nucleotide phosphodiesterase inhibitor. cAMP activates acAMP-dependent protein kinase, protein kinase A. As shown in FIG. 10,the reduction in serum-stimulated ³H-thymidine incorporation byAng-(1-7) or carbacyclin was completely blocked by pretreatment with theprotein kinase A inhibitor (PKAI) Rp-adenosine-3′,5′-cyclicmonphospho-phorothioate triethylamine salt (Rp-cAMPS) (Calbiochem, LaJolla Calif.). These results suggest that Ang-(1-7) is directly coupledto the Gs protein to activate adenylate cyclase and elevate cellularcAMP production. Alternatively, Ang-(1-7) may stimulate the productionof prostacyclin which binds to prostacyclin receptors coupled toadenylate cyclase and the synthesis of cAMP. Collectively, these resultssuggest that Ang-(1-7) causes an increase in the cellular levels of cAMPwhich stimulates the cAMP-dependent protein kinase to inhibit growth.

[0148] Ang-(1-7) may inhibit cell growth by preventing thephosphorylation and activation of MAP kinases in response to mitogenstimulation. For example, compounds that increase the intracellularconcentration of cAMP have been shown to reduce MAP kinase activity inVSMCs and fibroblasts and inhibit mitogen-stimulated growth in VSMCs(Cook, S. J. and McCormick, F., Science, 1993, 262:1069-1072; and Wu, J.et al., Science, 1993, 262;1065-1068). In addition, classic growthfactors, such as PDGF, epidermal growth factor, and basic fibroblastgrowth factor stimulate VSMC growth in vitro and in vivo. Growthstimulation by these mitogens as well as by Ang II is mediated, at leastin part, through activation of MAP kinases to induce early responsegenes and increase transcription.

[0149] The activity of the MAP kinases ERK1 and ERK2 in VSMCs wasmeasured using phospho-specific antibodies that only recognize theactivated protein kinases. Ang II caused a dose-dependent increase inboth ERK1 and ERK2 phosphorylation (37- and 166-fold increase overbasal), with maximal stimulation by 1 μM Ang II. Incubation of VSMCswith concentrations of Ang-(1-7) up to 1 PM had no effect on ERK1 orERK2 phosphorylation. However, pre-incubation with increasingconcentrations of Ang-(1-7) caused a dose-dependent reduction in AngII-stimulated ERK activity, with maximal inhibition at 1 μM Ang-(1-7).One micromolar Ang-(1-7) reduced 100 nM Ang II-stimulated ERK1 and ERK2activation by 42.3±6.2% and 41.2±4.2%, p<0.01, respectively, as shown inFIG. 11.

[0150] Ang-(1-7) also reduced ERK phosphorylation by 10 ng/mL PDGF inVSMCs, which increased ERK1 and ERK2 activities by 16-fold and 26-foldover basal, respectively. It was found that 1 μM Ang-(1-7) decreasedPDGF-stimulated ERK1 and ERK2 activities by 43.6±8.6% and 38.7±11.4%,p<0.05, respectively.

[0151] To begin to address the molecular mechanisms of Ang-(1-7)inhibition of the growth of cancer cells, MAP kinase activity wasmeasured in quiescent SK-LU-1 cells stimulated with 1% FBS, in thepresence and absence of increasing concentrations of Ang-(1-7). As shownin FIG. 12, Ang-(1-7) caused a dose-dependent decrease in the amount ofserum-stimulated ERK1 and ERK2 activities. These results suggest thatAng-(1-7) either inhibits the kinase which phosphorylates and activatesERKI and ERK2, a MAP kinase kinase, or stimulates the activity of a MAPkinase phosphatase, either of which would result in a decrease in activeMAP kinase.

[0152] Ang-(1-7) also inhibited platelet-derived growth factor (PDGF)-or epidermal growth factor (EGF)-stimulated ³H-thymidine incorporationinto human breast cancer cells of the ZR-75-1 cell line, as shown inFIG. 13. For these experiments, semi-confluent cell monolayers were madequiescent by a 48-h incubation in serum-free media, followed by a 28-htreatment period with increasing concentrations of Ang-(1-7) in thepresence of either 2.5 ng/mL PDGF or 100 ng/ml EGF. It was found thatAng-(1-7) reduces mitogen-stimulated human breast cancer cell growth ina dose-dependent manner.

[0153] The results show that Ang-(1-7) attenuates MAP kinase activationby either Ang II, serum, or growth factors PDGF or EGF, and thatAng-(1-7) can inhibit cell growth through a reduction in the activity ofmitogen-stimulated MAP kinases. Thus, Ang-(¹-7) may reduce MAP kinaseactivity by inhibiting the signaling pathways that stimulate MAP kinasephosphorylation or by stimulating MAP kinase phosphatase activity.

Example 8

[0154] Mechanisms of Inhibition of Cancer Cell Growth by Ang-(1-7)

[0155] To further assess transcriptional regulation involved in theinhibition of cancer cell growth and proliferation by Ang-(1-7), totalRNA isolated from SK-LU-1 cells treated with 1% serum in the presenceand absence of 100 nM Ang-(1-7) was analyzed using gene arrayhybridization. Cells were incubated for 2 or 8 h, and total RNA wasisolated using the Atlas Pure Total RNA Labeling System (ClontechLaboratories, Inc). The RNA concentration was quantified by UVspectroscopy and any degradation was assessed by ethidium bromidestaining intensity of 28S and 18S ribosomal RNA following agarose gelelectrophoresis. RNA isolated from seven different cell passages waspooled prior to gene array analysis to account for individualvariability in gene regulation. Radiolabeled cDNA, prepared from thepooled RNAs using the Atlas system, was incubated with DNase to degradeany residual DNA and then hybridized to the Human Cancer 1.2 Atlas CDNAExpression Array (Clonetech Laboratories, Inc). This gene array setcontains 1,176 characterized human cDNAs on positively-charged nylonmembranes. The resultant hybridization signals, visualized byphosphorimage analysis, were quantified using the computerized MCIDimaging system with gene array analysis software to identify potentialgene products which are up-regulated or down-regulated in response toAng-(1-7) stimulation.

[0156]FIG. 14 shows some of the results obtained by gene arrayhybridization. A number of genes involved in tumor suppression,apoptosis, and cell cycle inhibition were upregulated in SK-LU-1 cellstreated with Ang-(1-7), including the tumor suppressors p16^(INK4a) andmenin and genes encoding the proapoptotic proteins BAD and BAK as wellas apoptotic protease activating factor 1. In contrast, severaloncogenes, protein kinase and cell cycle progression genes weredownregulated. For example, MAP kinase kinase 5 (MEK5), ERK1, andp21/K-ras 2B were reduced, suggesting that Ang-(1-7) may alsochronically reduce the Ras/Raf/MEK/MAP kinase signaling cascade. Theseresults suggest a number of signaling pathways that may be involved inthe Ang-(1-7)-mediated reduction of cell proliferation observed in thelung cancer cells. Several candidate genes were selected forverification by RT-PCR and Western analysis.

[0157] The gene array hybridization results indicated that MAP kinasekinase 5 (MEK5) was downregulated in response to Ang-(¹-7). Thus, MEK5expression in SK-LU-1 lung cancer cells in response to Ang-(1-7) wasmeasured by RT-PCR and Western analysis. Quiescent SK-LU-1 cells werestimulated with 1% serum in the presence and absence of 10 nM Ang-(1-7).RNA was isolated using Trizol and whole cell lysates were isolated at 2,4, 8 and 24 h following treatment. As shown in FIG. 15, MEK5 mRNA andprotein were reduced 4 and 8 h following treatment with Ang-(1-7). Theresults of these experiments indicate that the cellular concentrationsof MEK5, a protein involved in MAP kinase signaling and cell growth, arereduced in human lung cancer cells following treatment with Ang-(1-7).In FIG. 15, intensities of RT-PCR products and protein bands weredetermined by image analysis. MEK5 protein levels increased immediatelyafter mitogen stimulation. Still, at both 4 and 8 hours, MEK5 mRNA andprotein levels are reduced by treatment with Ang-(1-7).

[0158] The gene array hybridization results also indicated that mRNAsencoding proteins that stimulate or participate in apoptosis (BAD, BAK,and APAF) are upregulated by Ang-(1-7) in mitogen-stimulated SK-LU-1cells. Stimulation of apoptosis by Ang-(I-7) was measured by generationof the caspase-3 cleavage product poly(ADP-ribose) polymerase (PARP), todetermine whether Ang-(1-7) stimulates apoptosis. Since casepase-3 isactivated during apoptosis, an increase in the generation of itscleavage product (PARP) is a measure of apoptosis. Cleaved PARP wasmeasured using an anti- cleaved product-specific antibody inserum-stimulated SK-LU-1 cells treated for either 2, 4, or 8 hours with10 nM Ang-(1-7). As shown in FIG. 16, an increase in the amount ofcleaved PARP was visualized following a 4 to 8 hour treatment withAng-(1-7), suggesting that Ang-(1-7) stimulates apoptosis. These resultssuggest that, in human lung cancer cells, Ang-(1-7) stimulates apoptosisto reduce cell growth.

[0159] The regulation of cell growth is a key element in the normalmaintenance of healthy tissue. A delicate balance exists between theproliferative and anti-proliferative factors controlling cell growth.The identification of the molecular mechanisms regulating cell growth isvital to understanding tumor formation, and the development ofanti-cancer therapeutices. In an embodiment, the present inventionrecognizes that Ang-(1-7), a peptide hormone present in the circulation,causes a marked decrease in cell proliferation of vascular cells as wellas cancer cell growth in vitro and in vivo. Ang-(1-7) is present in thecirculation at concentrations similar to the vasoconstrictor peptidehormone Ang II, and is generated from the precursor Ang I or Ang II bytissue peptidases and ACE inhibitors. Thus, in an embodiment, thepresent invention describes the use of a pharmaceutically effectiveamount of angiotensin-(1-7) or an angiotensin-(1-7) receptor agonist asa means to prevent tumor formation, or inhibit tumor growth in anindividual. The invention has been described in detail with particularreference to preferred embodiments thereof, but it will be understoodthat variations and modifications can be effected within the spirit andscope of the invention.

1 8 1 7 PRT Homo sapiens 1 Asp Arg Val Tyr Ile His Pro 1 5 2 8 PRTArtificial Sequence Synthetic peptide 2 Xaa Arg Val Tyr Ile His Pro Thr1 5 3 7 PRT Artificial Sequence Synthetic peptide 3 Asp Arg Val Tyr IleHis Xaa 1 5 4 10 PRT Homo sapiens 4 Asp Arg Val Tyr Ile His Pro Phe HisLeu 1 5 10 5 7 PRT Homo sapiens 5 Arg Val Tyr Ile His Pro Phe 1 5 6 6PRT Homo sapiens 6 Val Tyr Ile His Pro Phe 1 5 7 5 PRT Homo sapiens 7Val Tyr Ile His Pro 1 5 8 8 PRT Homo sapiens 8 Asp Arg Val Tyr Ile HisPro Phe 1 5

What is claimed is:
 1. A composition for inhibition of cell growth orproliferation comprising a pharmaceutically effective amount of anagonist for the angiotensin-(1-7) receptor in a pharmaceuticallyacceptable carrier, wherein a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist comprises an amount which issufficient to inhibit cell growth or proliferation.
 2. The compositionof claim 1, wherein said cells comprise cancer cells.
 3. The compositionof claim 2, wherein the cancer comprises bladder cancer, breast cancer,brain cancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.
 4. A composition for inhibition of cancer cellgrowth or proliferation comprising a pharmaceutically effective amountof an agonist for the angiotensin-(1-7) receptor in a pharmaceuticallyacceptable carrier, wherein a pharmaceutically effective amount ofangiotensin-(1-7) receptor agonist comprises an amount which issufficient to inhibit cancer cell growth or proliferation.
 5. Thecomposition of claim 4, wherein the cancer cells comprise a functionalangiotensin-(1-7) receptor.
 6. The composition of claim 4, wherein thecancer comprises bladder cancer, breast cancer, brain cancer, coloncancer, endometrial cancer, head and neck cancer, leukemia, lymphoma,lung cancer, melanoma, liver cancer, rectal cancer, ovarian cancer,prostate cancer, bone cancer, pancreatic cancer, skin cancer, or renalcancer.
 7. The composition of claim 4, wherein the cancer is in a humansubject.
 8. The composition of claim 4, wherein the angiotensin-(1-7)receptor agonist comprises angiotensin-(1-7) peptide having the sequenceset forth in SEQ ID NO:
 1. 9. The composition of claim 4, wherein theangiotensin-(1-7) receptor agonist is modified to increase its chemicalstability in vivo.
 10. The composition of claim 4, wherein theangiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions.
 11. The composition ofclaim 4, wherein the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.
 12. The composition of claim 4, further comprisinga compound which increases the efficacy or amount of angiotensin-(1-7)receptor agonist in the cancer.
 13. The composition of claim 12, whereinthe compound which increases the efficacy or amount of angiotensin-(1-7)receptor agonist increases angiotensin-(1-7) agonist synthesis.
 14. Thecomposition of claim 12, wherein the compound which increases theefficacy or amount of angiotensin-(1-7) receptor agonist decreasesangiotensin-(1-7) agonist degradation, metabolism or clearance.
 15. Thecomposition of claim 4, wherein a pharmaceutically effective amount ofsaid angiotensin-(1-7) receptor agonist increases cellularprostacyclins.
 16. The composition of claim 4, wherein apharmaceutically effective amount of said angiotensin-(1-7) receptoragonist increases cellular cAMP.
 17. The composition of claim 4, whereina pharmaceutically effective amount of angiotensin-(1-7) receptoragonist increases the expression of genes involved in tumor suppression,apoptosis, and/or cell cycle inhibition in the cancer.
 18. Thecomposition of claim 17, wherein the genes showing increased expressioncomprise BAD, oncostatin M-specific beta subunit, PDCD2, EGF responsefactor 1, CASP4, RBQ-3, p16-INK, menin, checkpoint suppressor 1, BAK,apoptotic protease activating factor-1, SOCS-3, insulin-like growthfactor binding protein 2, B-myb or the fau tumor suppressor.
 19. Thecomposition of claim 4, wherein said pharmaceutically effective amountof angiotensin-(1-7) receptor agonist decreases the levels of knownoncogenes, protein kinases, and/or cell cycle progression genes in thecancer.
 20. The composition of claim 19, wherein the genes showingdecreased expression comprise cell cycle entry regulator, ERK1, cellcycle progression 2 protein, p21/K-ras 2B oncogene, epithelial cellkinase, ser/thr kinase, MAP kinase kinase 5 (MEK5), beta catenin, ortyrosine-protein kinase receptor tyro3 precursor, protein phosphatase 2AB56-alpha, cyclin-dependent kinase regulatory subunit (CDC28), celldivision protein kinase 6 (CDK6), c-myc oncogene, ERBB-3 receptorprotein tyrosine kinase, A-kinase anchoring protein, or rho C.
 21. Thecomposition of claim 4, wherein the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-(1-7) receptoragonist at the cancer which ranges from 0.005 nM to 10 μM.
 22. Thecomposition of claim 4, wherein the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-(1-7) receptoragonist at the cancer which ranges from 0.05 nM to 1 μM.
 23. Thecomposition of claim 4, wherein the dose of angiotensin-(1-7) receptoragonist results in a local concentration of angiotensin-( 1-7) receptoragonist at the cancer which ranges from 1 nM to 100 nM.
 24. Acomposition to inhibit the growth or proliferation of cancer cells in anindividual comprising a pharmaceutically effective amount of a compoundwhich increases the efficacy or amount of circulating or cellularangiotensin-(1-7) receptor agonist in a pharmaceutical carrier, whereina pharmaceutically effective amount provides endogenous levels ofangiotensin-(1-7) receptor agonist which is sufficient to inhibit cancercell growth or proliferation.
 25. The composition of claim 24, whereinthe cancer comprises bladder cancer, breast cancer, brain cancer, coloncancer, endometrial cancer, head and neck cancer, leukemia, lymphoma,lung cancer, melanoma, liver cancer, rectal cancer, ovarian cancer,prostate cancer, bone cancer, pancreatic cancer, skin cancer or renalcancer.
 26. The composition of claim 24, wherein the compound whichincreases the efficacy or amount of circulating or cellularangiotensin-(1-7) receptor agonist increases angiotensin-(1-7) agonistsynthesis.
 27. The composition of claim 24, wherein the compound whichincreases the efficacy or amount of circulating or cellularangiotensin-(1-7) receptor agonist decreases angiotensin-(1-7) agonistdegradation, metabolism or clearance.
 28. The composition of claim 24,wherein the compound which increases the efficacy or amount ofcirculating or cellular angiotensin-(1-7) receptor agonist comprises anangiotensin receptor antagonist.
 29. A method to inhibit cell growth orproliferation comprising application of an agonist for theangiotensin-(1-7) receptor to said cells, wherein said cells have afunctional angiotensin-(1-7) receptor.
 30. The method of claim 29,wherein said cells comprise cancer cells.
 31. The method of claim 30,wherein the cancer comprises bladder cancer, breast cancer, braincancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.
 32. The method of claim 29, wherein the agonistfor the angiotensin-(1-7) receptor comprises angiotensin-(1-7) peptidehaving the sequence set forth in SEQ ID NO:
 1. 33. The method of claim29, wherein the angiotensin-(1-7) receptor agonist comprises a fragmentof angiotensin-(1-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions.
 34. The method ofclaim 29, wherein the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.
 35. A method to inhibit the growth or proliferationof cancer cells in an individual comprising application of apharmaceutically effective amount of a functional agonist for theangiotensin-(1-7) receptor to said individual, wherein apharmaceutically effective amount comprises sufficient angiotensin-(1-7)receptor agonist to inhibit growth or proliferation of the cancer cells.36. The method of claim 35, further comprising cancer cells having afunctional angiotensin-(1-7) receptor.
 37. The method of claim 35,wherein the cancer comprises bladder cancer, breast cancer, braincancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.
 38. The method of claim 35, wherein theangiotensin-(1-7) receptor agonist comprises angiotensin-(1-7) peptidehaving the sequence set forth in SEQ ID NO:
 1. 39. The method of claim35, wherein the angiotensin-(1-7) receptor agonist is modified toincrease its chemical stability in vivo.
 40. The method of claim 35,wherein the angiotensin-(1-7) receptor agonist comprises a fragment ofangiotensin-(i-7) or a functional equivalent of angiotensin-(1-7)comprising conservative amino acid substitutions.
 41. The method ofclaim 35, wherein the angiotensin-(1-7) receptor agonist comprises anon-peptide agonist.
 42. The method of claim 35, further comprisingapplication of a compound which increases the efficacy or amount ofcirculating or cellular angiotensin-(1-7) agonist.
 43. The method ofclaim 42, wherein the compound which increases the efficacy or amount ofangiotensin-(1-7) agonist decreases the degradation, metabolism orclearance of the angiotensin-(1-7) receptor agonist.
 44. The method ofclaim 42, wherein the compound which increases the efficacy or amount ofangiotensin-(1-7) agonist increases angiotensin-(1-7) synthesis.
 45. Themethod of claim 42, wherein the compound which increases the efficacy oramount of angiotensin-(1-7) agonist comprises an angiotensin receptorantagonist.
 46. The method of claim 35, wherein application of apharmaceutically effective amount of angiotensin-(1-7) receptor agonistin said individual increases cellular prostacyclins in said cancercells.
 47. The method of claim 35, wherein application of apharmaceutically effective amount of angiotensin-(1-7) orangiotensin-(1-7) receptor agonist in said individual increases cellularcAMP in said cancer cells.
 48. The method of claim 35, whereinapplication of a pharmaceutically effective amount of angiotensin-(1-7)receptor agonist increases the expression of genes involved in tumorsuppression, apoptosis, and/or cell cycle inhibition in the cancercells.
 49. The method of claim 48, wherein the genes showing increasedexpression comprise BAD, oncostatin M-specific beta subunit, PDCD2, EGFresponse factor 1, CASP4, RBQ-3, p16-INK, menin, checkpoint suppressor1, BAK, apoptotic protease activating factor-1, SOCS-3, insulin-likegrowth factor binding protein 2, B-myb or the fau tumor suppressor. 50.The method of claim 35, wherein application of a pharmaceuticallyeffective amount of angiotensin-(1-7) receptor agonist in saidindividual decreases the levels of known oncogenes, protein kinases,and/or cell cycle progression genes in the cancer cells.
 51. The methodof claim 50, wherein the genes showing decreased expression comprisecell cycle entry regulator, ERK1, cell cycle progression 2 protein,p21/K-ras 2B oncogene, epithelial cell kinase, ser/thr kinase, MAPkinase kinase 5 (MEK5), beta catenin, tyrosine-protein kinase receptortyro3 precursor, protein phosphatase 2A B56-alpha, cyclin-dependentkinase regulatory subunit (CDC28), cell division protein kinase 6(CDK6), c-myc oncogene, ERBB-3 receptor protein tyrosine kinase,A-kinase anchoring protein, or rho C.
 52. The method of claim 35,wherein the dose of angiotensin-(1-7) receptor agonist results in alocal concentration of angiotensin-(1-7) receptor agonist at the cancerwhich ranges from 0.005 nM to 10 μM.
 53. The method of claim 35, whereinthe dose of angiotensin-(1-7) receptor agonist results in a localconcentration of angiotensin-(1-7) receptor agonist at the cancer whichranges from 0.05 nM to 1 μM.
 54. The method of claim 35, wherein thedose of angiotensin-(1-7) receptor agonist results in a localconcentration of angiotensin-(1-7) receptor agonist at the cancer whichranges from 1 nM to 100 nM.
 55. A method to inhibit the growth orproliferation of cancer cells in an individual comprising application tosaid individual of a pharmaceutically effective amount of a compoundwhich increases the efficacy or amount of circulating or cellularangiotensin-(1-7) receptor agonist in said individual.
 56. The method ofclaim 55, wherein the cancer comprises bladder cancer, breast cancer,brain cancer, colon cancer, endometrial cancer, head and neck cancer,leukemia, lymphoma, lung cancer, melanoma, liver cancer, rectal cancer,ovarian cancer, prostate cancer, renal cancer, bone cancer, pancreaticcancer or skin cancer.
 57. The method of claim 55, wherein the compoundwhich increases the efficacy or amount of cellular angiotensin-(1-7)agonist increases angiotensin-(1-7) synthesis.
 58. The method of claim55, wherein the compound which increases the efficacy or amount ofcellular angiotensin-(1-7) agonist decreases angiotensin-(1-7) agonistdegradation, metabolism or clearance.
 59. The method of claim 55,wherein the compound which increases the efficacy or amount ofcirculating or cellular angiotensin-(1-7) agonist comprises an Ang IIangiotensin receptor antagonist.
 60. A kit for inhibiting cancer cellgrowth or proliferation in an individual comprising: (a) at least onecontainer comprising a pharmaceutically effective amount of a functionalagonist for the angiotensin-(1-7) receptor, wherein a pharmaceuticallyeffective amount comprises an amount of angiotensin-(1-7) receptoragonist which is sufficient to inhibit cancer cell growth orproliferation; (b) a pharmaceutically acceptable carrier; and (c)instructions for use.
 61. The kit of claim 60, further comprising apharmaceutically effective amount of a compound which increases theefficacy or amount of circulating or cellular angiotensin-(1-7) agonist.62. The kit of claim 60, wherein the cancer cells comprise a functionalangiotensin-(1-7) receptor.
 63. The kit of claim 60, wherein the cancercomprises bladder cancer, breast cancer, brain cancer, colon cancer,endometrial cancer, head and neck cancer, leukemia, lymphoma, lungcancer, melanoma, liver cancer, rectal cancer, ovarian cancer, prostatecancer, bone cancer, pancreatic cancer, skin cancer, or renal cancer.64. The kit of claim 60, wherein the angiotensin-(1-7) receptor agonistcomprises angiotensin-(1-7) peptide having the sequence set forth in SEQID NO:
 1. 65. The kit of claim 60, wherein the angiotensin-(1-7)receptor agonist is modified to increase its chemical stability in vivo.66. The kit of claim 60, wherein the angiotensin-(1-7) receptor agonistcomprises a fragment of angiotensin-(1-7) or a functional equivalent ofangiotensin-(1-7) comprising conservative amino acid substitutions. 67.The kit of claim 60, wherein the angiotensin-(1-7) receptor agonistcomprises a non-peptide agonist.